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11.11.2015

Biologists John Herr and Eusebio Pires in the lab where they first discovered the surprising link between the surfaces of growing eggs and tumor cells.
University of Virginia School of Medicine researchers have discovered a new strategy for attacking cancer cells that could fundamentally alter the way doctors treat and prevent the deadly disease. Herr and his research partner, Department of Obstetrics and Gynecology biologist Eusebio Pires, both specialize in germ cells a€“ the reproductive cells that make up sperm and eggs. At the time, Herr and Pires were studying a protein called SAS1B that is typically only on the surface of developing and mature egg cells. The restriction of SAS1B to growing eggs suggests strategies for developing improved female contraceptives that selectively target only the pool of growing eggs, potentially reducing unwanted side effects of current steroidal contraceptives. Live human uterine cancer cells stained green at cell surface locations where an antibody has attached to the cell surface protein SAS1B.
He and Pires have found a way to exploit this fundamental insight by developing a method for delivering medication using the SAS1B protein as a target. Since the SAS1B protein appears only on egg cells and cancer cells, the molecule can serve as a target for tiny tracking probes created using monoclonal antibodies. After about an hour, the antibody-SAS1B complexes reach compartments inside the cell and release their toxic drug payload, triggering changes leading to cell death within a few days. This kind of targeted drug delivery could mean a dramatic reduction in the difficult side-effects of traditional cancer treatment such as hair loss, nausea, anemia and neuropathy.
While the monoclonal antibodies would have to attack the pool of growing egg cells in addition to the SAS1B positive cancer cells, the ovaries' supply of dormant eggs would remain healthy and untouched by the treatment. In addition to treating cancer, these selective antibodies could also lead to a new method for early cancer detection and prevention. Pires and Herr hope that doctors will one day be able to use the monoclonal antibodies to measure patients' SAS1B protein levels in blood.
Cancer cells riddled with holes and dying after treatment with an antibody drug conjugate that targets the SAS1B protein on the surface of tumor cells.
The researchers' findings have been published in the scientific journal Oncotarget in an article written by Eusebio S. Monoclonal antibodies directed against tumor antigens have proven effective for treating some forms of cancer.
Researchers at the University of Michigan have described a new approach to discovering potential cancer treatments that requires a fraction of the time needed for more traditional methods. A diet that starves triple-negative breast cancer cells of an essential nutrient primes the cancer cells to be more easily killed by a targeted antibody treatment, UW Carbone Cancer Center scientists report in a recent publication. New research reveals that infection with the Epstein-Barr virus (EBV) may put some women at increased risk for developing breast cancer. If you walked into a cancer clinic ten years ago as a newly diagnosed patient, you'd likely get "standard of care" treatment based on the location of the cancer in your body and its stage. Oxford University researchers have found a way to detect ovarian cancer early and identified an enzyme that is key in making ovarian cancer more deadly. Most cancer drugs are designed to halt cell growth, the hallmark of cancer, and one popular target is the pathway that controls the production of a cell's thousands of proteins. AbstractImage guided technique is playing an increasingly important role in the investigation of the biodistribution and pharmacokinetics of drugs or drug delivery systems in various diseases, especially cancers.
The properties of biodistribution and pharmacokinetics play a major role in influencing and determining the efficacy and safety for the treatment with a medicine.
Magnetic resonance imaging (MRI) is a non-invasive medical imaging to visualize detailed internal structures. 3.1 Radiolabeling of antibody therapeutics and principle of PET imagingPET imaging is another kind of non-invasive option for diagnosis.
3.2 PET images guided biodistribution and pharmacokinetic study of theranosticsTargeted therapeutic and diagnostic nanocarriers functionalized with antibodies, peptides or other targeting ligands that recognize over-expressed receptors or antigens on tumor cells have potential in the diagnosis and therapy of cancer. Simple radioimmuno-nanoparticles diagram of a BNF-type particle coated with PEG to link 111In-ChL6. Imaging modalities such as CT and PET have been applied sequentially in the diagnosis and staging of disease and in monitoring the effects of therapy for several years.
Optical imaging is a photonic technique which applied in the area of imaging, describing the behavior of visible, ultraviolet, and infrared light used in imaging [62, 63]. The blood-brain barrier (BBB) is a special tight capillaries membrane in CNS that formed from a continuous layer of endothelial cells bound together with tight junctions that allow very little transcellular or pericellular transport of molecules in blood fluid [99].
Film autoradiography of Rhesus monkey brain removed 2 h after an intravenous administration of [125I]-humanized HIRMAb. Molecular imaging takes advantage of the traditional diagnostic imaging techniques and introduces molecular imaging probes to measure the expression of indicative molecular markers at different stages of diseases. Corresponding author: Hong Ding, Department of Immunology, Florida International University, Miami, FL33199.
Cancer therapy has been characterized throughout history by ups and downs, not only due to the ineffectiveness of treatments and side effects, but also by hope and the reality of complete remission and cure in many cases. Cetuximab-resistant cells are sensitive to the EGFR tyrosine kinase inhibitor gefitinib and the monoclonal antibody to EGFR panitumumab. Figure 1: Cetuximab-resistant cells are sensitive to the EGFR tyrosine kinase inhibitor gefitinib and the monoclonal antibody to EGFR panitumumab.
We addressed this challenge through a new physical concept that exploited (i) the difference in the membrane electric properties between the tumor and healthy cells and (ii) the capability of magneto-electric nanoparticles (MENs) to serve as nanosized converters of remote magnetic field energy into the MENs' intrinsic electric field energy.
By more selectively targeting cancer cells, this method offers a strategy to reduce the length of and physical toll associated with current treatments. While researching new methods of contraception, Pires and Herr discovered a surprising link between developing egg cells and tumors. So far, they have found SAS1B expressed in breast, melanoma, uterine, renal, ovarian, head and neck, and pancreatic cancers.
Monoclonal antibodies are highly pure antibodies designed to bind to one single target protein with a uniform affinity. Both women and men can use the treatment, which is predicted to dramatically limit unwanted side effects on healthy normal cells.
This means normal ovulation could begin again once treatment is complete and oocytes are again recruited to develop and ovulate; a process anticipated to take approximately 200 days.
Pires explained that the research team is developing a way to find tiny amounts of free-circulating SAS1B proteins in the body.
Those with elevated levels of the protein would be tested for the early stages of cancer and receive treatment sooner. Despite the increasing use of monoclonal antibody therapy, it is not clear how these antibodies drive tumor removal. Department of Immunology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL33199.2. Besides anatomical imaging modalities such as computed tomography (CT), magnetic resonance imaging (MRI), molecular imaging strategy including optical imaging, positron emission tomography (PET) and single-photon emission computed tomography (SPECT) will facilitate the localization and quantization of radioisotope or optical probe labeled nanoparticle delivery systems in the category of theranostics. Currently, several image guided modalities have been applied in biomedicine and even in clinic, including magnetic resonance imaging (MRI), X-ray computed tomography (CT), positron emission tomography (PET), single-photon emission computed tomography (SPECT), electron microscopy, autoradiography, optical imaging and ultrasound (US), etc. Since biodistribution of antibody-based therapeutics is a key consideration that can be modulated to impact the ensuing in vivo pharmacological effect. SPECT images guided biodistribution and pharmacokinetics study of theranosticsSingle-photon emission computed tomography (SPECT) is a nuclear medicine tomographic imaging technique using gamma rays.
Although PET offers high sensitivity in terms of molecular concentrations and it is possible to measure picomolar amounts of a PET radiopharmaceutical in a tissue. These optical techniques, such as bioluminescence and fluorescence, are emerging as powerful new modalities for molecular imaging in disease diagnosis and therapy. Such nanomaterials have their advantages over traditional fluorescent probes such as organic dyes and fluorescent proteins [79, 83]. The BBB is an obstacle for clinicians and complicates the treatment of diseases of the central nervous system (CNS). It has been widely reported the importance and advantages of various imaging modelities in the drug development. Within the therapeutic arsenal, alongside surgery in the case of solid tumors, are the antitumor drugs and radiation that have been the treatment of choice in some instances. IntroductionChemotherapy, surgery and radiotherapy are the most common types of cancer treatments available nowadays. Cell viability was measured after treating DiFi parental cells and DCR clones with increasing concentrations of cetuximab for 72 h. This capability allows to remotely control the membrane electric fields and consequently trigger high-specificity drug uptake through creation of localized nano-electroporation sites. That link may allow doctors to use antibodies to deliver medication directly to tumors while sparing healthy tissue.
For female cancer patients especially, a drug that doesn't touch their body's reserve of quiescent eggs could be a huge breakthrough. Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, New York 14260.
They are all noninvasive imaging modalities and proven clinical applications, and some of them are only extended to in-vivo research specimens as small as mice [1, 2]. After being labeled with radionuclide of sufficient long half-life, antibody therapeutics can be monitored using PET (positron emission tomography) imaging with respect to pharmacokinetics and its biodistribution.
Biodistribution and pharmacokinetics could also be recorded by blood sampling at predetermined time points, preferably associated with radiometabolite evaluation with respect to time [23].
With a three dimentional images obtained by PET, establishing a ROI (region of interest) and set the location of the organs or tissues in the images is of critical importance. Optical imaging offers higher sensitivity and temporal resolution than PET at the same spatial resolution in small animals, but unlike PET is limited to a few centimeters of tissue depth [35].
For example, they have robust photochemical stability, high quantum yield, and excellent resistance to chemical and photochemical degradation, as well as size-tunable photoluminescence that ranges from visible to near-IR with sharp spectral bands [84]. In order to develop BBB associated theranostic techniques, neuroimaging techniques have become increasingly important in assessing the biologic and physiologic properties of brain tumors and neurologic lesions (11). We summarized the distribution and pharmacokinetics studies using MR imaging, CT, PET imaging, SPECT imaging, optical imaging as well as SPECT etc. In recent years, immunotherapy has become an important therapeutic alternative, and is now the first choice in many cases. The history of chemotherapy began in the early 20th century, but its use in treating cancer began in the 1930s. In in-vitro studies on human ovarian carcinoma cell (SKOV-3) and healthy cell (HOMEC) lines, we applied a 30-Oe d.c. Then the monoclonal antibody-SAS1B complexes can function as tiny injectors for targeted medication. They are recruited by the immune system to identify and neutralize foreign objects like bacteria and viruses. Among them, PET and optical imaging are regarded as quantitative or semi-quantitative imaging modalities that employ radiotracers or optical tracers to image biodistribution of the labeled drugs or probe loaded delivery systems in the body, meanwhile CT and MRI are normally used for anatomical imaging purposes [3, 4]. This causes the nuclei to produce a rotating magnetic field detectable by the scanner, which can be recorded to construct an image of the body. Normally, it is assumed that the labeling method does not perturb the property of the molecules with respect to metabolism, specific binding or non-specific interactions. After being administered in vivo, the gamma-emission of the isotope from the radiolabeled nanoparticles or antibody allows the drug to be seen by a gamma-camera.Since the source of SPECT images are gamma ray emissions, the radioisotope decays and emits gamma rays, which can be detected by a gamma camera to obtain 3D images, which permit accurate localization of organs [33].
The bioluminescence and fluorescence can be obtained from dyes, specific metal materials, and biological resources etc and already used in diagnosis and therapy in research and clinic [64-66]. Also, QDs with different emission colors can be simultaneously excited with a single light source, with minimal spectral overlap, providing significant advantages for multiplexed detection of molecular targets [85, 86].
Although there are many successful imaging platforms including X-ray tomography (CT), optical imaging, positron emission tomography (PET), single-photon-emission computed tomography (SPECT), ultrasound (US), and magnetic resonance imaging (MRI), etc. Among those imaging modalities, PET, SPECT and MRI imaging has been used in clinical study, while optical imaging is still on the way of getting through; almost all of the studies using optical imaging are in the preclinical status. Nanotechnology has recently arrived on the scene, offering nanostructures as new therapeutic alternatives for controlled drug delivery, for combining imaging and treatment, applying hyperthermia, and providing directed target therapy, among others. The term “chemotherapy” was coined by the German scientist Paul Ehrlich, who had a particular interest in alkylating agents and who came up with the term to describe the chemical treatment of disease. Cell lysates from DiFi cells and DCRs were collected after 2 h of treatment with cetuximab.
In these imaging modalities, non-invasive technique attracted more attention because of its properties of no breaking in the skin and no contact with the mucosa, internal body cavity beyond a natural or artificial body orifice. Strong magnetic field gradients cause nuclei at different locations to rotate at different speeds, thus providing 3D spatial information.
This information is typically presented as cross-sectional slices through the patient, but can be freely reformatted or manipulated as required. Indeed, in many cases anatomic imaging is used exclusively, although functional imaging with PET is fulfilling an increasingly important role in the staging and therapy monitoring processes, particularly when the CT scan is equivocal.
With the help of combing of innovative molecular biology and chemistry, scientists have developed optical methods for imaging a variety of cellular and molecular processes in vivo, including the receptor-ligand interaction, tumor targeting, bio-distribution of proteins such as antibody, etc.
Thus, with conjugation with antibody and drug on the same QDs, this kind of theranostic reagent can be used as new type of probes for optical bioimaging and treatment on cancer.Although there are significant advantages of semi-conductor nanocrystal QDs in the application of optical imaging for cancer detection, the potential toxicity cannot be ignored due to the existing of Cadmium element component inside the particles.
MRI and combining with US seems the most successful applications in the clinic.Nanoparticles of course can be employed as effective tool for theranostic purpose. Non-invasive detection of various molecular markers of diseases can allow for much earlier diagnosis, earlier treatment, and better prognosis that will eventually lead to personalized medicine.
These therapies can be applied either alone or in combination with other components (antibodies, peptides, folic acid, etc.). During the First and Second World Wars, it was noticed that soldiers exposed to mustard gas experienced decreased levels of leukocytes.
The drug penetrated through the membrane and completely eradicated the tumor within 24 hours without affecting the normal cells.IntroductionThe development of a technology that is capable of high-specificity targeted delivery of anti-neoplastic drugs would be a significant breakthrough in cancer in general and ovarian cancer in particular.
Non-invasive in vivo molecular imaging can be obtained from PET, MR, CT and visible infrared in vivo optical imaging systems. Comparing with other medical imaging techniques such as computed tomography (CT), MRI provides good contrast between the different soft tissues of the body, which make it especially useful in imaging brain, muscles, the heart, and cancers [14, 15]. The results showed that PET blood curves agreed well with direct measurements within 12% at all time points.
Therefore, it can be used to provide information about localized function in internal organs, such as functional cardiac or brain imaging.
Whereas optical imaging has been used primarily for research in small-animal models, in some specific area optical imaging may have the possibility to translate into clinical medicine. In order to alleviate possible toxicity and increase the stability of QDs in biological field, Ding et al successfully employed carboxyl functional Pluronics F127 (F127COOH) into the antibody conjugated QDs for pancreatic cancer detection and obtained satisfied results.
One biocompatible stealth nanoparticles were designed by using microemulsion methodology to optimize BBB circumvention and encapsulate drugs for brain delivery.
For using molecular imaging to investigate the biodistribution and PK properties of theranostics, the following aspects still need further investigation:Major advantages of radionuclide-based molecular imaging techniques (SPECT and PET) over other modalities (optical and MRI) are high sensitivity (picomolar level), quantitation, and non-tissue penetration limitation. This led to the use of nitrogen mustard as the first chemotherapy agent to treat lymphomas, a treatment used by Gilman in 1943.
Although the circulatory system can deliver a drug to every cell in the body, delivering the drug specifically inside the tumor cell past its membrane without affecting the healthy cells remains a challenge1,2,3. The discovery of the first modern non-invasive techniques was setup at the end of 19th century based on physical methods: electrocardiography and X-rays. Another advantage of MR imaging is that it does not need to use ionizing radiation which has damage to cell structure once be exposed.X-Rays computed tomography, also called computed tomography (CT scan) or computed axial tomography (CAT scan), can make use of radiation to get an internal view of the body for medical imaging employing tomography created by computer processing.
Among them, Copper-64 (64Cu) is a promising PET radionuclide, particularly for antibody-targeted imaging [20]. The 4.2-day half-life of 124I favors its use for PET study of monoclonal antibodies (mAbs).
For example, 3D acquisition SPECT of the liver was performed to investigate the correlation between tumor accumulation of In-111-bevacizumab and VEGF-A expression in patients with colorectal liver metastases [34].Although heart has been used as an organ in PET imaging to estimate blood concentration, for pharmacokinetic study using SPECT imaging, the commonly used method is to collect blood samples from patients or animals at timed intervals after injection of the radioisotope-labeled mAb [23]. This device provides a medical imaging department with the capability to acquire accurately aligned anatomic and functional images for a patient from a single scanning session [47]. The advantages of optical imaging for assessing antibody pharmacokinetics in vivo are the conjugation chemistry, cost, and high-speed throughput. They successfully applied anti-mesothelin antibody in vivo for pancreatic cancer detection [87]. In vitro and in vivo data showed that the formulation has no effect on BBB barrier integrity and membrane permeability, or alteration of expression of the BBB tight-junction proteins, occludin, and claudin-1 while crossing BBB [100]. However, one disadvantage is that the resolution of either SPECT or PET is not as high as MRI.
Here, we present a review of the evolution of cancer treatments, starting with chemotherapy, surgery, radiation and immunotherapy, and moving on to the most promising cutting-edge therapies (gene therapy and nanomedicine). In the following years, alkylating drugs such as cyclophosphamide and chlorambucil were synthesized to fight cancer [1,2]. In ovarian cancer, intraperitoneal (IP) delivery through a surgically implanted catheter has shown improved survival rates. From that time, non-invasive with advantage of penetrating the body rather than a scalpel have continuously enlarged the scope of medical technology.
Although CT technique is regarded as birth defects or DNA altering because of powerful X-ray radiation exposure, it is still largely employed in hospitals, especially used for broken bones and skulls. The reported methods for modification include addition of labeled groups or radiolabel chelating groups. The major advantages of radionuclide-based molecular imaging techniques (SPECT and PET) over other modalities (e.g. The fluorophore conjugation chemistry is often carried out with a commercially available kit and can be performed in most laboratories without special license, equipment, hazardous precautions, or specific training. The in vitro and in vivo toxicity study showed the low damage to cells and tissues in small animals.
We offer an historical point of view that covers the arrival of these therapies to clinical practice and the market, and the promises and challenges they present. Kilte and Farber designed folate antagonists such as aminopterin and amethopterin, leading to the development of methotrexate, which in 1948 achieved leukemia remission in children [3]. Parental DiFi and DCR cells were treated with increasing concentrations of gefitinib, and viable cells were measured after 72 h. However, catheter complications and toxicity have precluded widespread adoption of this invasive means of delivery4.
Non-invasive techniques commonly can be used for the purpose of theranostics by diagnostic imaging and therapy on diseases [5-7].The ability to quantitatively image the biodistribution of therapeutics or drug delivery systems in a noninvasive manner can aid in the development of new theranostic, dose optimization and treatment monitoring. Not like MRI imaging which can provide subtle differences between the different kinds of soft tissues, CT can demonstrate the difference between bone density and soft tissue. Compared with radio-halogenation, radiometal ion complexation reactions have simpler chemistry and allow tracer production kits.
Although from the ROI of heart on the PET images, the blood concentrations can be calculated, most of the case, the pharmacokinetic study was done by withdrawing blood sample and analyzing them with radioassay or ELISA [24].The distribution of monoclonal antibodies is determined by the rate of extravasation in tissue, the rate of distribution within tissue, binding affinity in tissue and the rates of elimination from tissue, route of injection, species etc. The detection systems are generally 30 to 50% as expensive as those for small-animal SPECT or PET.
Another alternative on optical imaging is using biodegradable polymer encapsulated NIR dye for cancer detection [88].
Nanoparticles encapsulation or conjugation of transporter substrates have already been used for enhance the access of drugs to the brain. CT is preferable in the application of bones imaging while MRI is good for tissue imaging.For both PET imaging and optical imaging, the image only showed the signals from the radioisotope probe or fluorophore probe, while the probe may still attach to the metabolite of drug even after the degradation of the drug. Elion and Hitchings developed 6-thioquanine and 6-mercaptopurine in 1951 for treating leukemia [4,5]. Current research attempts to go around these limiting factors by using nanoscale systems5,6,7. Normally, the information of biodistribution can often be obtained by dissecting the animal, collecting plasma or tissues and being analyzed by high-performance liquid chromatography (HPLC), Enzyme-linked immunosorbent assay (ELISA) etc [8-10], the key advantage of non-invasive imaging is less time consuming and more cost effective of animals and analysis reagents [11]. Although MRI and X-ray are both imaging techniques for organs of the body, the difference in application is that MRI images provide a 3D representation of organs, which X-Rays usually cannot.Dr.
For example, chelator 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) is commonly used in radiolabeling copper radioisotope with antibody [21].'MicroPET' is a small-animal positron emission tomography which uses pharmacologically or biochemically active compounds labeled with short-lived or long-lived positron-emitting radionuclides to monitor their biodistribution, physiological processes and biochemical pathways etc [22].
However, one disadvantage is that the resolution (typically >1 mm) of either SPECT or PET is not as high as the other imaging modalities such as MRI.
Planar fluorescence images are generated within seconds or minutes, typically with a photograph overlay.
With proper fluorescent dye or drug encapsulation, this kind of biodegradable copolymer can be conjugated with target ligands or specific antibody for all purpose of tumor targeting. In last decade, nano-sized carrier comprised by polymer, emulsion, liposome, nanocrystals, micelles and etc, are already make great progress in pharmaceutical field for drug targeting and imaging. Heidelberger developed a drug for solid tumors, 5-fluorouracil (5-FU), which is up to now an important chemotherapy agent against colorectal, head and neck cancer [6].
Cells were cultured for 2 h with gefitinib, and cell lysates were subjected to western blot analysis using antibodies to the indicated proteins. Often, as immunological reagents, monoclonal antibodies are used to recognize the tumor-specific biomarker while the nanoscale control further improves the specificity and targeted drug delivery capability in general8,9,10. When the key is inserted into the lock, the antibody activates, tagging or neutralizing its target.
There is a critical need to establish an effective non-invasive tool to clearly diagnose diseases along with the treatment. These radionuclides are incorporated into molecules during the radiosynthesis step, generating the radiotracer. The movement of antibody into tissue by convective transport is thought to be the primary mechanism for the distribution in tissue [28]. Optical imaging experiments also can accommodate four to five animals per scan, allowing an efficient researcher to perform 100+ animal images per hour, whereas PET and SPECT imaging is often limited to one to two animals per 10 to 30 min scan. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size exclusion HPLC for confirming the stability of the labeled antibody therapeutics may be a good way of providing more information of metabolism.
The 1950s saw the design of corticosteroids, along with the establishment of the Cancer Chemotherapy National Service Center in 1955, whose purpose was to test cancer drugs. Nonetheless, in spite of the tremendous progress in this field during the last decades, the capability of targeted delivery with adequately high specificity (to tumor cells) remains an important roadblock to finding a cure for cancer.In this paper, we present a study in which we address this challenge through a new physical concept. If a high effective non-invasive method exists, a cancer patient could be more potentially received effective treatment at an earlier stage with concise targeted drug delivery [12].Therefore, discussions of perspectives of various imaging guided modalities are focusing on the research applications in biodistribution and pharmacokinetics of delivery systems such as theranostics. By MRI images, they found out the rapid blood-pool uptake (5-9 hours) of mAb after intraperitoneal injection followed by tumor localization (26-32 hours).
The transport is mainly determined by the movement rate of fluid from blood to tissue and by the sieving effect of paracellular pores in the vascular endothelium.
Radiolabeling of nanoparticles was also applied for PK and biodistribution studies for the purpose of theranostics. The larger antibody formats (IgG and miniantibody) gave higher tumor uptake levels than did the smaller formats (diabody and scFv). Such throughput and cost combined with expected improvements in quantification from advances in instrumentation provide an exciting outlook for this preclinical fluorescence imaging approach [67].A key challenge for pharmacists and clinic doctors is defining the disposition of drugs such as monoclonal antibody and pharmacokinetic property in whole animals or patients. This kind of luminescent probe can even used for sentinel lymph node mapping since it is one of the most important site that the metastatic happened [91]. Ding and his colleagues applied inorganic gold nanords for siRNA delivery into brain has been succeeded in vitro and in vivo based on their contrast property in cancer detection [64, 101-103]. For labeled antibody, due to its therapeutic effect and detective property, the labeled antibody can also be considered as theranostics if it is used in the field of treatment and diagnosis.After obtaining biodistribution data using molecular imaging in preclinical study, establishing physiologically based pharmacokinetic model (PBPK) and allometric scaling for translation of animal data to human clinical pharmacology is very important. At that time, monotherapy drugs only achieved brief responses in some types of cancers [7]. Parental DiFi and DCR cells were treated with increasing concentrations of panitumumab, and viable cells were measured after 72 h.
It exploits (i) the difference in the electric properties of the membrane between the tumor and healthy cells and (ii) the ability of the recently discovered body-temperature magneto-electric nanoparticles (MENs) to function as nano-converters of remotely supplied magnetic field energy into the MENs' intrinsic electric field energy11,12,13. And micro-PET finding was consistent with the data from high resolution MRI images slices for mAb bio-distribution in main organs of tumor-beard mice [16].
The radiotracer accumulates in the tissues, and its radionuclide decays by emission of a positron (anti-electron). Normally, the biodistribution of drugs requires sacrificing a number of rodents and quantitating the tissue concentration by HPLC, ELISA or by counting the radioactivity emitting from the tissue.
Ongoing discoveries of these potential target molecules in disease also drive pharmaceutical and biotechnology companies to find leading agents that selectively regulate these molecular pathways in vitro and in vivo. Although the above nanomaterials have their advantages over traditional fluorescent dye probes, batch to batch reproducibility and quantization of the labeled drug still need further investigation. They also applied fluorescent nanocrystal as theranostic carrier for CNS drug delivery system [104].
By 1958, the first cancer to be cured with chemotherapy, choriocarcinoma, was reported [8]. Like the conventional magnetic nanoparticles (MNs), MENs have a non-zero magnetic moment and therefore can be controlled remotely via application of an external magnetic field. Theranotics is defined as a material that combines the modalities of therapy and diagnostic imaging at the same time within the same dose [13]. After travelling at most a few millimeters, a positron will collide with an electron, simultaneously releasing two gamma rays (photons) with energy of 511 keV into opposite directions.
Helbok et al radiolabeled DTPA-derivatized lipid-based nanoparticles including including (111)In and (99m)Tc for SPECT and PET images with therapy in vitro and in vivo.
The smaller formats were rapidly cleared from circulation, and the diabody, which accumulated in the tumor, may be more suitable for radiodiagnostic applications. Among all the existing techniques, optical imaging including bioluminescence and fluorescence, are emerging as key techniques to meet these challenges and advance molecular imaging in preclinical research and patient care. Whether the biodistribution of the labeled therapeutic antibodies is significantly affected by the nanomaterials remains a question to be addressed.Over the past years, different kinds of optical imaging techniques have been developed for biomedical applications, including various microscopy methods for in vitro and ex vivo applications as well as several methods for in vivo applications such as bioluminescence imaging, fluorescence imaging, ultrasound, dark field, diffused optical tomography, and optical coherence tomography, etc.
Various imaging modalities mentioned in this review are useful quantitative tools for investigating the above biopharmaceutical properties of theranostis. Cell lysates from DiFi cells and DCRs were collected after treatment for 2 h with panitumumab. However, unlike MNs, MENs offer a new far-reaching function, which is an energy-efficient control of the intrinsic electric fields within the nanoparticles by an external magnetic field. Teng et al conjugated anti-mouse OxLDL polyclonal antibody to polyethylene glycol-coated ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, and imaged at 7.0 Tesla MRI on a carotid perivascular collar model in atherosclerotic disease mice. These two photons are detected by the PET camera and by collecting a statistically significant number of radioactive events, mathematical algorithms reconstruct a three-dimensional image that shows the distribution of the positron-emitting molecules in the tissues [19].


Their study showed that PEGylated DTPA-nanoparticles were suitable for targeting applications. The combination of PET with CT provides easier localization of tissues [34, 48].PET is sensitive in the process of detecting and quantifying a low abundance molecular target in cells while MRI is capable of providing exquisite anatomy for organs. Bioluminescence is the process of light emission in living organisms, it is a chemical process wherein light is emitted during the interaction of a protein produced in engineered cells and an administered substrate, for example firefly luciferase and luciferin [68].
Not only with help from expertise from studies on materials, various techniques can be used in combination, either simultaneously or sequentially, to provide complementary information from the same cells, tissues, organs, or animals [92-96].
Koffie et al prepared biodegradable nanocarrier systems made up of poly(n-butylcyanoacrylate) dextran polymers coated with polysorbate 80 (PBCA nanoparticles) to deliver BBB-impermeable molecular imaging probes into the brain for targeted molecular neuroimaging. Better treatments were developed, with alkaloids from vinca and ibenzmethyzin (procarbazine) applied to leukemia and Hodgkin's disease [9-11]. This unprecedented capability is a result of the strong magneto-electric (ME) coupling in this new class of nanostructures even at body temperature11,12,13.
The final goal of the theranostic is to donate materials with the capacity of monitoring the treated tissue, PK and efficacy in the long-term period. The results showed MRI signal loss in the carotid atherosclerotic lesions after administration of targeted anti-OxLDL-USPIO at 8th hour and 24th hour, which means that anti-mouse OxLDL antibody conjugated magnetic nanoparticle can be used as a strategy for the therapeutic evaluation of atherosclerotic plaques in vivo by MRI imaging [17].CT became an important tool in medical imaging to supplement X-rays and medical ultrasonography since 1970s.
Single-photon emission computed tomography (SPECT) is also one kind of nuclear medicine tomographic imaging technique using gamma rays which can provide 3D information as PET as well. Biodistribution data as measured by gamma counting were consistent with the PET findings [11]. Biodistribution in Lewis rats revealed no significant differences between nanoparticles in terms of DTPA loading and particle composition; however, different uptake patterns were found between the radionuclides used [37]. Bioluminescence imaging is a technology developed over the past decade that allows for the noninvasive study of ongo-ing biological processes in small laboratory animals.
In the 1970s, advanced Hodgkin's disease was made curable with chemotherapy using the MOMP protocol [12,13], which combined nitrogen mustard with vincristine, methotrexate and prednisone, and the MOPP protocol [14,15], containing procarbazine but no methotrexate.
As a result, MENs introduced in a biological microenvironment act as localized magnetic-to-electric-field nano-converters that allow remote control and generation of the electric signals that underlie the intrinsic molecular interactions. This approach will benefit nanomedicine with an effectively style of personalized treatment.
Non-invasive PET imaging of vascular endothelial growth factor (VEGF) expression using radiolabeled bevacizumab has been investigated in ovarian cancer models [29]. Quantitative values from PET of a large number of biological parameters are complemented by the high-resolution information provided by MRI (in the micromolar range) to yield complementary information.
Bioluminescence imaging utilizes native light emission from one of several organisms which contain bioluminescence.
Chen's lab applied fluorescent Maestro imaging system by real-time video imaging of protease expression in vivo, they found out that PEGylation of a molecular beacon with a specific size PEG can significantly alter the activation properties of the probe targeting matrix metalloproteinases (MMPs). PBCA nanoparticles do not induce nonspecific BBB disruption, but collaborate with plasma apolipoprotein E to facilitate BBB crossing [105]. Patients with diffuse large B-cell lymphoma were treated with the same therapy and, in 1975, a cure for advanced diffuse large B-cell lymphoma was reported using protocol C-MOPP, which substituted cyclophosphamide for nitrogen mustard [16].Surgery and radiotherapy were the basis for solid tumor treatment into the 1960s.
DiFi and DCR cells were treated with cetuximab or panitumumab for 2 h and stimulated with EGF for 15 min.
In actual applciations, the combination of MRI, CT, PET, or optical imaging and their combination usage may add further impetus to the rapid and high throughput screening of nanoparticle delivery systems based diagnosis and therapeutics (Figure 1). After being administered in vivo, the gamma-emission of the isotope from the radiolabeled materials allows the drug to be seen by a gamma-camera. Small-animal PET and microCT images were used to calculate tumor uptake and compared with ex vivo biodistribution at 168 h after injection. An MRI-based method has the potential to improve PET image quality, increasing its reliability, reproducibility, and quantitative accuracy [49].
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation with a different wavelength. And MMPs are a family of zinc-dependent endopeptidases and represent the most well-known proteases associated with tumorigenesis [97].Among them, both fluorescence and bioluminescence imaging techniques have been found wide applications for in vivo tumor optical imaging in mouse models and afford convenient, frequent visualization and measurement of tumor biomarkers in a real time, sensitive, and noninvasive way.
Some representative applications of imaging modalities in the area of biodistribution and pharmacokinetics of nanoparticles for the aim of treatment and diagnosis are also listed in this review.
The radiation dose for a particular study depends on multiple factors: volume scanned, patient build, number and type of scan sequences, and desired resolution and image quality. PET data and MRI data have been combined for monitoring the drug anticancer effect [50], the response of patients to antibody treatment [51] as well as for detection of lymph node metastases [52], which is very important for the improvement of tumor detection and for the guide of mAb-based therapy.Overall, accurate quantization of PET tracer uptake levels in tumors is important for staging and monitoring response to treatment. It also can allow for longitudinal detection of disease progression and therapeutic response in the same animals so as to minimize the subject-to-subject variability and reduce the animal number required by a traditional method. Qiao et al conjugated lactoferrin (Lf) with poly(ethylene glycol) (PEG)-coated Fe3O4 nanoparticle to cross BBB by receptor-mediated transcytosis via the Lf receptor present on cerebral endothelial cells.
There were some promising publications about the use of adjuvant chemotherapy after radiotherapy or surgery in curing patients with advanced cancer. Normally, the high resolution and image quality means the high exposure of radiation to patients. Therefore, it can be used to provide information about localized function in internal organs, such as functional cardiac or brain imaging [11]. Inductively heating the nanoparticle by externally applied alternating magnetic field caused tumor necrosis after 24 h of therapy.
The combination with anatomical information obtained from CT will improve quantization of tumor values in terms of bias and variance [53].
Fluorescence imaging uses probes that emit light (fluorescence) after the excitation at a fluorophore-specific wavelength. Importantly, such in vivo studies are very valuable for bridging the gap between in vitro and in vivo studies and facilitating preclinical and further translational studies [98]. The MRI images confirmed that in vivo experiments, Fe3O4-Lf probe exhibited an enhanced effective measure for delivering the nanoparticles across the BBB [106, 107].It is well-known that few of those antibodies can penetrate the shield of tightly packed cells known as BBB. Breast cancer was the first type of disease in which positive results with adjuvant therapy were obtained, and also the first example of multimodality treatment, a strategy currently employed for treatment of numerous types of tumors. In this study, we exploit this capability to achieve the field-controlled specificity of the drug-loaded MENs as required to significantly improve the state of chemotherapy.The MEN's new capability to control the local electric fields remotely (via magnetic fields) opens an exciting and previously unexplored path to exploit the intrinsic electric properties of the cell membrane. The high ionizing radiation in CT application can damage the DNA double strand structure in human body. This project demonstrated the quantization of radio-labeled bevacizumab in tumor using non-invasive imaging is convenient, which was also validated by ex vivo biodistribution studies.
Using appropriate software to fuse PET and CT image together and accurately quantify the tumor uptake level of tracer is of critical value.
In most cases, emitted light has a longer wavelength, and therefore lower energy, than the exciting source, known as the Stokes shift [69]. Normally, antibody concentrations in the brain are typically about a thousand times lower than in the blood.
In the late 1960s, the use of adjuvant chemotherapy changed the concept of localized treatment.There was significant progress in 1978 when higher cure rates of metastatic germ cancer were achieved by combining cisplatin, bleomycin and vinblastine [17-19]. Due to the presence of ion channels and other electric-field driven properties, the cell membrane is an electrically polarizable medium. In one study, researchers found out at a typical CT scanning, 40%-100% of the irradiated cells is damaged by one or more double strand breaks in DNA molecule. Since VEGF is a major angiogenic factor responsible for the development of tumor vasculature, the expression level of VEGF could be regarded as a factor indicating disease progress. A delay in tumor growth occurred after the alternating magnetic field treatment, which was statistically significant when compared with the untreated group [38, 39]. The experience with polychemotherapy in hematologic cancer brought to light the fact that different drugs act against tumor cells in different phases of their cellular cycle. Although there is repair process in cells for damaged and broken DNA, the results are not satisfied [18].
PET imaging were performed in mice bearing human colorectal cancer (HT29) xenografts to investigate the in vivo biodistribution of VEGF after injection of 64Cu-DOTA-bevacizumab.
Wu and colleagues applied NIR dye labeled Bevacizumab antibody to find out the pharmacokinetics, lymph Node Uptake and mechanistic PK behavior model after i.v.
One of these solid tumor drugs was CMF (cytoxan, methotrexate and fluorouracil), a standard therapy for treating breast cancer for over 30 years. In fact, electroporation is one such well-known characteristic that exploits the dependence of the membrane's porosity on the electric field16,17,18,19,20,21. The specific region on an antigen that an antibody recognizes and binds to is called the epitope, or antigenic determinant.
That's why by now, CT technique is designed to combine with other image techniques for better application in patients. Misri and colleagues also applied SPECT imaging for mesothelin expressing in tumors by evaluation of 111In labeled antibodies. The electroporation has been widely studied as a means to trigger drug delivery into the cells. Their findings indicated that the antimesothelin antibody mAbMB may be developed into a diagnostic agent for imaging mesothelin-expressing cancers [40].
Significant correlation was obtained between the organ-to-muscle ratios measured by the radioactivity and fluorescence intensity [59]. As a consequence, numerous drugs with various mechanisms of action were introduced during the 1980s. By clinging to this receptor, the antibody is transported into the brain, where it can act against β-secretase 1. Subsequent advances and developments led to liposomal therapy, which places drugs inside liposomes (vesicles made of lipid bilayers), decreasing some of the side effects of chemotherapy such as cardiotoxicity. Our new approach was to use MENs to exploit the promising delivery technique by scaling it down into the nanoscale. The chain of amino acids does not exist in a 2 dimensional structure but appears as a 3 dimensional structure. Wu et al also successfully applied gamma scintigraphy by pulsincap capsule in the gastrointestinal tract of dogs by to find out the desintegration and transit behavior of Tetramethylpyrazine phosphate. To assess the biophysical characteristics of this novel nanosystem, the HSA coated IONPs (HSA-IONPs) were dually labeled with 64Cu-DOTA and Cy5.5, and tested in a subcutaneous U87MG xenograft mouse model. This finding may provide quite useful aim for the future protein biodistribution and pharmacokinetic studies.The most important application of optical imaging in the biological field is early cancer detection or diagnosis in preclinic and clinic studies, they provided a very useful alternative modality for tumor imaging [73]. Examples of liposomal drugs include liposomal doxorubicin and daunorubicin, one of the first steps in nanotechnology-based approaches. Due to this NANO-ELECTROPORATION, magnetic-field-activated MENs loaded with the drug and optionally with the biomarker-specific antibodies (for delivery to the tumor cells) can generate localized fields large enough to open up the membrane pores in their proximity only and thus let the drug inside the tumor cells.
An epitope may only be recognized in its form as it exists in solution, or its native 3D form. Nano-graphene oxide (GO) sheets with covalently linked, amino group-terminated six-arm branched polyethylene glycol (PEG; 10 kDa) chains were conjugated to NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid, for (66)Ga-labeling) and TRC105 (an antibody that binds to CD105).
Their SPECT images confirmed the time controlled capsule releasing in the gastrointestinal delivery in dog at the expected time schedule [41]. Murine monoclonal antibodies to the rat transferrin receptor, such as the OX26 monoclonal antibody, are targeted through the BBB on the transferrin receptor in the rat. The 1990s sparked the beginning of targeted chemotherapy by screening for specific critical molecular targets.
Because this process is relatively energy efficient, most of the energy goes to fulfill the main operation (of opening up the local pores, i.e. The capillary depletion technique demonstrated transcytosis of the RI7-217 rat monoclonal antibodies antibody can through the mouse BBB in vivo. These advances in modern chemotherapy and studies on genetics and molecular biology contributed to the ongoing decline in death rates. The orthotopic gliomas beared mice was subcutaneous injected with AF680-labeled 1D11, a pan-neutralizing TGF-β antibody, and used for the bio-distribution study via in vivo imaging and fluorescence microscopy.
The studies indicate rat monoclonal antibodies to the mouse transferrin receptor may be used for brain drug-targeting studies [110]. Data from the genome sequence suggested that many dysfunctions associated with cancer could be due to the abnormal function of some protein kinases.
The interaction between the MENs and the electric system of the membrane effectively serves as a field-controlled gate to let the drug-loaded nanoparticles enter specifically the tumor cells only.
In 4T1 tumor-bearing mice, these GO conjugates were primarily cleared through the hepatobiliary pathway. Madru's work, Nanoparticles with a solid iron oxide core and a polyethylene glycol coating were labeled with (99m)Tc. Besides the monoimaging principle, combining two or more different imaging modalities in the same agent can be of considerable value in molecular imaging. Boado and his colleagues used engineered murine monoclonal antibody to the human insulin receptor (HIR) as a brain drug delivery system for transport across the human BBB (Figure 4).
The first tumors targeted with drugs approved by the FDA (Food and Drug Administration) and the EMEA (European Medicines Agency) were renal cell cancer, hepatocellular cancer and gastrointestinal stromal tumors.
PET imaging successful demonstrated the in vivo tumor targeting property with GO, showing them to be suitable nanoplatforms for future biomedical research such as cancer theranostics [31].Andresen TL et al covalently attached octreotate (TATE) to the distal end of DSPE-PEG(2000) on PEGylated liposomes with an encapsulated positron emitter (64)Cu that can be utilized for PET imaging. By binding a biotinylated anti-Her2 Herceptin antibody to provide tumor targeting, a biotinylated DOTA chelator labeled with 111In and a biotinylated Cy5.5 fluorophore to a streptavidin nanoparticle, the imaging results show high tumor accumulation and strong tumor-to-normal tissue contrast by both fluorescence and nuclear imaging.
The HIRMAb was humanized by complementarity determining region (CDR) grafting on the framework regions (FR) of the human B43 IgG heavy chain and the human REI kappa light chain. In recent years, numerous specific tumors have been tested with various kinase inhibitors and there is a trend towards combining chemotherapy with these new targeted therapies.Chemotherapy is curative in some types of advanced cancer, including acute lymphoblastic and acute myelogenous leukemia, Hodgkin's and non–Hodgkin's lymphoma, germ cell cancer, small cell lung cancer, ovarian cancer and choriocarcinoma. TATE are somatostatin peptide analogs, can target Somatostatin receptors (SSTRs) that are over-expressed in neuroendocrine tumors (NETs).
The humanized HIRMAb avidly bound to the HIR of isolated human brain capillaries, which are used as an in vitro model system of the human BBB.
In pediatric patients, curable cancers include acute leukemia, Burkitt's lymphoma, Wilms' tumor and embryonal rhabdomyosarcoma. First, the biomarker-specific antibodies steer the drug-loaded MENs (to which they also are attached) to the tumor cell membrane. They used PET imaging to investigate the biodistribution and pharmacokinetics of the (64)Cu-loaded PEGylated liposomes with and without TATE. In Cohen's work, IRDye800CW was coupled with monoclonal antibodies of cetuximab and bevacizumab with 89Zr multi-labeling for optical imaging of tumor targets. The humanized HIRMAb was radiolabeled with 125-iodine, and injected intravenously into an adult, anesthetized Rhesus monkey.
Although treatment is not always curative for these cancers, there has been significant improvement in progression-free and overall survival. Second, even higher specificity is achieved due to the fact that the tumor and healthy cells have different values of the threshold field, Hth, for the “gate” to open up. They found that the presence of TATE on the liposomes resulted in a significantly faster initial blood clearance in comparison to control-liposomes without TATE. Digital autoradiography images revealed a nonhomogeneous distribution of (99m)Tc- labeled superparamagnetic iron oxide nanoparticles within the lymph nodes; nanoparticles were found in the cortical, subcapsular, and medullary sinuses [61]. The biodistribution studies showed that blood clearance was enhanced and live uptake was increased at 24 to 72 hours post injection when 2 or more eq of dye had been coupled to mAbs. Brain scanning showed the humanized HIRMAb was rapidly transported into all parts of the primate brain after intravenous administration. Another modality of treatment is neoadjuvant therapy, which aims to reduce the size of the primary tumor and prevent micrometastases.
Indeed, it is well-known that the electric properties differ significantly between the healthy and tumor cells of the same type22. This study realized that after labeling with IRDye800CW, optical imaging can be used for accurate quantification of the biodistribution of mAbs (Figure 2) [76].
The humanized HIRMAb may be used as a brain drug and gene delivery system for the targeting of large molecule therapeutics across the BBB in humans [111]. This type of treatment improves on more conservative surgical techniques in preserving the functionality of important organs.
In general, the tumor cells have substantially lower values of the potential compared to that of the healthy cells. Neoadjuvant chemotherapy is indicated for anal, breast, lung, gastroesophageal, rectal, bladder and head and neck cancer, as well as some types of sarcoma.
Consequently, the cancer cells must also have a significantly lower value of the threshold field for the drug-loaded MENs to enter the cell. There are many cancers for which adjuvant chemotherapy has been established with curative effect, and with the new effective drugs and combinations the curability rates are expected to rise even more. The results showed that (64)Cu-loaded PEGylated liposomes with TATE conjugated to the surface could be promising new imaging agents for visualizing tumor tissue and especially NETs [32]. Since 1990, the incidence and mortality of cancer have been declining and despite the increase in the elderly population [22], mortality rates for the United States declined from 2005 to 2007.In 1890, Halsted performed the first radical mastectomy, believing that cancer would be more curable if surgical techniques were more aggressive, thus avoiding regional recurrences.
Moreover, the same order of magnitude electric can be generated by much smaller magnetic fields, of the order of 100?Oe, if one takes into account the edge effects because of the cubic symmetry of the real-life nanoparticles, as shown below. He had many followers at that time, but thanks to advances in chemotherapy, radiotherapy, biology and technology, the outlook now is quite different. Due to the edge effect (significantly enhanced charge density at the edges), the electric field in the vicinity of MENs could be amplified by a factor of 100 or more depending on the proximity to the edge. Radical surgery has now been replaced by less extensive operations.The turn of the 20th century marked the beginning of the development of cancer surgery techniques, with the first abdominoperineal resection performed in 1908 by Miles [23], the first lobectomy being performed in 1912 [24,25] and the first radical hysterectomy performed by Wertheim in 1906, all carried out under oncological criteria. Moreover, the edge effects in the case of ME materials might be even further enhanced because both the electric charge density and the effective magnetic charge density are amplified at the edges.
Ideally, after the drug-loaded MENs penetrate into the cell cytosol through the “open” pores in the membrane, the drug can be released off the MENs by further increasing the field above the second critical value, Hr, necessary for overcoming the drug-MEN binding energy. Modern surgery has changed significantly, with Halstedian techniques replaced by non-invasive procedures such as laparoscopic colectomy (for the removal of colon cancer) [26], videothoracoscopy, radiofrequency ablation and radiosurgery techniques such as Cyberknife® [27]. We described one underlying hypothesis of this field-controlled drug release process in a recent article14. Breast-conserving surgery with sentinel-node removal has been used to improve esthetic results and avoid lymphedema [28].
This field strongly depends on the binding force between the MEN and the drug and consequently can be tuned in a large range through using different intermediate coating materials, field excitation frequencies and treatment durations. Another example of conservative surgery is the use of laryngoscopic laser surgery in early laryngeal cancer [29].
In summary, according to our idealistic hypothesis, there are two critical field values, Hth and Hr, that define the drug penetration threshold through the tumor cell membrane and the following release of the drug into the cell cytosol, respectively.
The most recent development is the Da Vinci®, a robotic system for the removal of cancer from prostate and kidney [30].The discovery of X-rays and radiation by Becquerel and Rontgen in the late 19th century was the first step towards radiation treatment. To ensure the required specificity of the uptake to the cancer cells only, the external applied field, HA, needs to be higher than the release field for the tumor cells, Hr_cancer, and lower than the threshold field for the healthy cells, Hth_healthy. In summary, using MENs not only can provide field-controlled delivery but also can significantly improve the specificity to tumor (compared to the specificity defined by the monoclonal antibodies alone). After World War II, technological progress allowed charged particles to be propelled through a vacuum tunnel called linac, or linear accelerator. In 1960, Ginzton and Kaplan began to use a rotational linac radiotherapy called “Clinac 6”, which was used to concentrate X-rays more deeply thereby they not affecting the skin as much. The development of modern computers enabled three-dimensional X-ray therapy, such as intensity-modulated radiation therapy (IMRT) using mapping information from Computed Tomography (CT) scans. When two or more antigen binding sites are identical, an antibody can form a stronger bond with the antigen.
This provides a three-dimensional reconstruction, which helps avoid toxicity since the contours of the tumor are targeted and separated from healthy tissues. Cytoreductive surgery followed by chemotherapy with mitotic inhibitor Paclitaxel (PTX) with platinum is the gold standard in treating EOC.
This treatment uses CT-guided IMRT technology that directs the radiation source by rotating it around the patient, which makes the morphological limits of a tumor easier to trace with the beam [31]. Another significant trend is the use of charged particle radiotherapy with proton or helium ions for specific types of patients with melanoma of the uveal tract.
It is also used as adjuvant therapy for skull base chondroma, chondrosarcoma and spine (usually cervical). As noted, there are technical considerations and limitations to IP therapy, although it is more effective than IV therapy. In summary, the lines of development have been fractionated dose delivery, technological advances in X-ray production and delivery and improvement of computer-based treatment planning.The latest advance in scanning technology with radiotherapy therapy is four-dimensional (4D) conformal radiotherapy [32], which records a video sequence of tumor movement.
In either case, the specificity of the drug uptake is still relatively low and as a result EOC remains a highly lethal malignancy. This therapy uses dynamic CT images of the body that compensate for any movement by the target, including movements when patients breathe. In addition, because of the high-specificity capability, the new nanotechnology can be used for targeted treatment of both localized and metastasized tumor cells.
Lower doses of radiation are used along with a biological agent, and stimulation by radiation produces cytotoxic agents. Light chain types are based on differences in the amino acid sequence in the constant region of the light chain. This complex technology was developed to use radiation to activate promoters and thus inducing the expression of genes responsible for producing enzymes. These proteins activate the selected drug, and the activated form of the drug then destroys cancer cells.
Through kinetics studies we confirmed that the drug penetrated through the tumor cell membrane and eradicated the majority of the cells within a 24-hour period without affecting the surrounding healthy cells. Another modality consists of radiolabeled molecules, which fight cancer by delivering targeted radiation to specific receptor-bearing cells. Radioactive isotopes (Iodine-125 or Indium-111) emit Auger electrons, which have the potential to be delivered to specific sets of target cells, thus sparing healthy cells.This manuscript reviews the evolution of oncological treatments available today, together with several immunotherapeutic approaches and nanoscale-based therapeutics including successes, drawbacks and recent progress.
A comparative analysis of the effect of the intermediate layer type on Hr is summarized in Fig.
These are the most versatile immunoglobulins and can carry out all functions of Ig molecules. ImmunotherapyThe concept of Immunotherapy in medicine incorporates the use of components of the immune system, including antibodies (Abs), cytokines, and dendritic cells, to treat various illnesses, such as cancer, allergies, and autoimmune and infectious diseases. Immunotherapy also includes the use of vaccines for the prevention of allergies and tumors.
By default, in order to provide adequate coupling between the MENs and Flutax-2 (to provide the initial release field of the order of 30?Oe), before being loaded with the drug, the MENs were coated with 3-Angstrom thick glycerol monooleate (GMO) layers. The zeta-potential and size of the MENs, GMO-MENs, HER2-GMO-MENs, and PTX-GMO-MENs are shown is Table S3.
Of all the types of anti-tumoral immunotherapy, this review will focus on the use of antibodies, their history, problems and current applications. For the purpose of a comparative analysis, we studied the following combinations of nanoparticles: (i) MENs loaded with PTX, (2) MENs loaded with PTX and the popular cancer biomarker HER-2 antibody, (3) free PTX, and (4) conventional MNs loaded with PTX. As the conventional MNs, 30-nm magnetite nanoparticles were used.Field-controlled drug release by MEN-based carriersDrug release from these different MEN-based combinations was triggered by a magnetic field at different strengths and frequencies, according to the physics described in our earlier paper on the release of ARV drug AZTTP for treatment of HIV-1 virus in the brain14. Antibodies: HistoryAntibodies (Abs) are one of the most important defense mechanisms for vertebrate animals. The pellet obtained after the drug loading procedure was washed thrice with the phosphate-buffered saline (PBS) buffer, to remove any residual unbounded drug. They are produced by B cells, which, after antigen-mediated activation, undergo differentiation to secretory (plasma) cells thus producing soluble antibodies. The drug-loaded-MENs' pellet was added to 1?ml of the PBS buffer in a vial and subjected to a d.c. Antibodies are highly specific, and they recognize and eliminate pathogens and disease antigens, but can be deliberately generated to recognize different target molecules (tumor markers, bacteria, receptors, cytokines, hormones, etc.). Thus, Abs can be used in many applications, including diagnostic techniques, research and therapy (against infections, tumors, transplants and autoimmune diseases).Antibodies were described in 1890 (Figure 1) by von Behring and Kitasato as “anti-toxins” that appeared in the serum of animals after immunization with inactivated toxins (toxoids) [33]. The researchers noted that protection could be transferred to other animals through the use of these antisera, thus beginning what it is known as “serum therapy” for treating infectious diseases (diphtheria and tetanus) in humans. Soon after, these sera elements were described as “anti-bodies” because they could be directed not only against toxins, but also against a large variety of organisms and compounds (bacteria, proteins, chemicals, etc.). Immunotherapy initially began with the use of antisera obtained from animals such as horses and sheep containing, among other things, a mixture of antibodies from the activation of different B cell clones, so-called “polyclonal antibodies” (PAbs). After exposing the vial to any magnetic field environment under study, the supernatant was obtained by spinning the sample at 3,000?rpm for 5 minutes and at 10°C.
In 1926, Felton and Bailey obtained pure antibodies, but it was not until the 1960s, thanks to the work of Porter and Edelman (1972 Nobel Prize winners), that the Ab structure became known. The supernatant was measured for the amount of the released drug spectrophotometerically through the absorbance at the PTX maximum wavelength of 230?nm30.The results of the field-controlled drug release spectrophotometry (absorption) experiments are summarized in Fig. After the introduction of Abs to therapy, researchers observed that the transferred defense was only temporary (as opposed to vaccination, which induces long-term memory). In addition, it often incurred anaphylactic responses that were occasionally fatal and which greatly reduced their use in human therapy.
However, these problems did not prevent PAbs from being used successfully in diagnostictechniques and even in preventive therapies. 2A shows the percentage of the drug release after a 1-minute exposure to a magnetic field at three strengths, 12, 44, and 66?Oe, respectively, for three different frequencies, 0, 100, and 1000?Hz, respectively.
Anti-snake venom, ant-tetanus and anti-Rh+ gamma globulins are still being used in clinical practice.In 1975, Cesar Milstein and George Kohler (1984 Nobel Prize winners) succeeded in generating monoclonal antibodies (mAbs) by fusing mouse B cells with B cell tumors (myeloma) to create hybrid cells, which were immortal and had the capacity to produce large quantities of a single (monoclonal) antibody [34]. As expected (see explanation above), for each frequency, there was a critical field, Hr, at which the drug release was significantly boosted. In 1976, genetic studies by Susumu Tonegawa revealed the basis for the vast diversity of antibodies, identifying the process of somatic recombination in immunoglobulin genes [35]. The increase of the frequency in the range up to 1000?Hz under study increased the release efficacy (by over 40%) especially at the low field range. Since the publication of the monoclonal antibody technique, mouse and rat mAbs have been used in many laboratories with thousands of applications in various scientific fields, in diagnostic techniques (clinical, food, environmental), research and in therapy (antitumor, autoimmune diseases). Monoclonal antibodies have helped in the discovery of new molecules (such as the identification of more than 300 membrane proteins, grouped under the CD concept or Cluster of Differentiation), transcription factors, viral, plant and bacterial proteins, phosphorylated compounds involved in death by apoptosis, factors involved in enzymatic cascades and many more. 2B illustrates the kinetics of the field-strength-frequency dependence of the release for the five values of the field exposure times, 1, 5, 10, 60, and 120 minutes, respectively.
It fixes with complements and is a good agglutinating Ig that leads to elimination of microbes. As an example of their usefulness, the current classification of leukemia by the World Health Organization is based on the presence or absence of membrane molecules recognized by monoclonal antibodies that define leucocyte populations in various stages of differentiation.But one of the greatest achievements with monoclonal antibodies is their use in human therapy.


Surgery, chemotherapy and radiotherapy are not specifically directed to tumor cells and may also affect healthy tissue.
For every exposure time setting, a fresh solution with PTX-loaded GMO-coated MENs was used. Antibodies can provide specificity and lower toxicity, opening new therapeutic possibilities. The field-triggered drug release was also confirmed through atomic force microscopy (AFM), Fourier Transform Infra-Red (FTIR), mass spectrometry, and X-ray diffraction (XRD) pattern studies. The first evidence of this potential came in 1982 when a patient suffering from lymphoma responded to treatment using a mouse mAb directed specifically against his tumor B lymphocytes [36]. This response rapidly encouraged research into the production of potentially therapeutic Abs. However, clinical trials results revealed that many patients receiving this therapy developed an immune response directed against the therapeutic Abs, a response known as HAMA (Human Anti-Mouse Antibodies) or HARA (Human Anti-Rat Antibodies). In contrast to mouse or rat myeloma cells, human myeloma cells proved difficult to adapt to continuous growth in vitro. Researchers tried to resolve this problem by immortalizing B cells using the Epstein-Barr virus (EBV) [37] and by fusing human B cells with well-established murine myeloma (obtaining heterohybridomas) [38]. However, the low production of antibodies in these cells, the instability of heteromyeloma cells and numerous technical problems lead to the search for alternative methods for generating human-like mAbs in the mid-1980s. These results showed that the drug uptake increased by a factor of five for the drug carried by field-controlled MENs compared to the drug driven by the HER-2 antibodies. Rituximab, a chimeric anti-CD20 mAb, was the first mAb approved by the FDA for antitumor therapy.
However, a year earlier, several mAbs conjugated with radioactive elements were approved for in vivo tumor detection. The HOMEC cells were cultured according to the same procedures that are described for the SKOV-3 cells in Section Methods.
Every year since then, several mAbs have been approved for therapy in the US and Europe, and more than half of them are chimeric or humanized mAbs (See Table 1).In addition to fully engineered antibodies, antibody fragments also have advantages compared to whole antibodies, especially in terms of the rate of solid tumor penetration. As a control, the cells with GMO-MENs only (without Flutax-2) were treated under the equivalent conditions. Jainr [39] determined that an intact IgG molecule needed 54 hours to move 1 mm into a solid tumor, whereas a Fab fragment reached the same distance in 16 hours.
The cell culture plates with the MENs and drug-GMO-MENs were exposed to three different field strengths, 5, 15, and 30?Oe, respectively. These radiolabelled antibodies are used for diagnosis or detection of whole cells, receptors and enzymes.
While the expression of chimeric and humanized antibodies was carried out in eukaryotic hosts, such as mammalian or plant cells, bacteria have been the most widely used organism for the production of recombinant antibody fragments [40-42].
However, despite numerous advantages, such as avoiding animal immunization and hybridoma production, their low cost and easier production [43], antibody fragments have shorter circulating half-lives compared to full-size antibodies, lack glycosylation and lack effector functions due to the absence of their Fc region (unless added). The measurements showed that as the field was increased above approximately 30?Oe, the drug penetration into the cancer cells (SKOV-3) greatly increased.
Modified versions, such as PEGylation of fragments (modification of a molecule by linking of one or more polyethylene glycol chains) [44] to improve circulation half-life, glycosylation and Fc region engineering are some of the recent approaches used by researchers to overcome these problems [45].In the mid 1990s, thanks to the development of molecular biology techniques and microinjection and manipulation of embryonic cells, several groups created various transgenic mice models carryinghuman Igs genes (Figure 1). This changed with the advent of monoclonal antibodies, as described in 1975 by KA¶hler and Milstein. The introduction of human Ig loci in these mice was carried out using various vectors, such as miniloci, yeast and human artificial chromosomes (YACs and HACS, respectively) and P1 vectors.
Transgenic mice can be immunized with almost any Ag (including human tumor cells), and their spleens can be used to obtain hybridomas following the conventional protocol [46-49]. The three key combinations of the carrier included (i) no particle, (ii) HER-2-GMO-MENs (Note: Here, HER-2 stands for the HER-2 biomarker antibody), and (iii) GMO-MENs, respectively.
Fully human monoclonal antibodies show several advantages in human therapy, which include low or no immunogenicity, better interaction with human effector systems and patterns of glycosylation and a longer half-life in human serum. 6A show the morphology of the cancer cells after 24-hour treatment by (i) the free drug (with no particle carrier), (ii) drug-HER-2-GMO-MENs with no field applied, and (iii) drug-GMO-MENs in a 30-Oe d.c. Examples of these new reagents include antibody alternative protein scaffolds based on leucine-rich repeat molecules of lamprey variable lymphocyte receptors (VLRs), libraries of fibronectin domains and designed ankryin repeat proteins (DARPs) [50]. With all these novel antibody formats, immunogenicity, stability and aggregation problems should be carefully considered.
6B show the morphology of the cancer cells after the 24-hour treatment by the same three combinations of the carrier with no drug present. This uses many new monoclonal antibodies against different cell-surface structures and helps flow cytometry in diagnosis if blood cancers.
In addition, we conducted the confocal imaging and the trypan-blue cell viability tests on both SKOV-3 and HOMEC cell lines after 24- and 36-hour field treatment. Flow cytometry has also more recently been used in the monitoring of disease and in the evaluation of tumor response to therapy. However, when it came to the field of human therapy, pharmaceutical companies did not initially show much interest in the development of monoclonal antibodies, although several research groups were showing promising results in preclinical and clinical studies. The reasons for their reluctance are many:A number of pharmaceutical companies had experience with generating small compounds, most of them chemically synthesized, but not with generating large biological molecules produced by cells. Moreover, sophisticated equipment and cell culturing under controlled conditions, with full quality assurance, are necessary for antibody production.There was the perception by pharmaceutical companies that production of mAbs was not going to yield sufficient profit. The assay is based on the reduction of XTT tetrazolium salt by the viable cells to form orange colored formazan derivative. Most companies preferred to concentrate their efforts on developing analogues of well-known drugs rather than on new products, while at the same time most clinicians opted for trials using combinations of known agents. In this assay, 1 ? 105 cells were seeded per well in a 96-well plate and incubated at 37°C for 24 hours. Advances in mAb engineering helped develop more effective mAb drugs with high specificity, improved potency and stability and decreased immunogenicity, which helped change the companies' initial reluctance.In terms of clinical trials, there were concerns about the cost of the trials (around 10 times more expensive today than 30 years ago), the time required for preclinical pharmacology and toxicology studies (which are much more regulated) and the difficulty in conducting early clinical trials. Then, the cell medium was replaced with a fresh one and washed with the PBS buffer and cell viability assay was performed by adding 50??l per well of XTT-activated solution from the XTT test kit supplied by ATCC and incubate for 4 hours at 37°C.
Since new drugs can only be tested against advanced and usually heavily pretreated disease, it is unlikely that dramatic responses will occur with these patients.The requirement for fetal calf serum in cell hybridoma cultures introduced another problem when Mad Cow Disease was identified in the early 1990s. The FDA proposed a limit on materials used in some medical products in order to keep them free of the agent thought to responsible for Mad Cow Disease (also known as bovine spongiform encephalopathy or BSE), making it necessary to find alternatives, such as enriched media without serum.
S7, no significant cytotoxicity was observed.Heat-dissipation due to field-treatment with MENsIn this experiment, the temperature was measured locally via Infra-red (IR) camera FLIR-i3 on the surface of both cancer (SKOV-3) and healthy (HOMEC) ovarian cells before and after a field treatment, as shown in Fig. Current Antibodies Used in Cancer TherapySince 1988, 228 mAbs have entered clinical studies for various diseases, with 56% of those currently in clinical development. Used to prevent acute rejection of transplanted kidneys and in T cell lymphoma Rituximab binds to CD20 and used in Non Hodgkin’s lymphoma Heceptin a€“ used in metastatic breast cancer Antibodies during and before childbirth Rhesus factor, also known as Rhesus D (RhD) antigen, is an antigen found on red blood cells. Presence of the antigen makes a person Rhesus-positive (Rh+) and absence makes a person Rhesus-negative (Rha€“). The first mAb approved for cancer therapy was rituximab (Rituxan™), a chimeric antibody directed against CD20, for non-Hodgkin's lymphomas. During normal childbirth, delivery trauma or complications during pregnancy, blood from a fetus can enter the mother's system.
Since then, many others have reached the market, including those for the treatment of breast cancer (trastuzumab, Herceptin®), acute myeloid leukemia (gemtuzumab Ozogamicin, Mylotarg™), chronic lymphocytic leukemia (alemtuzumab, Campath-1H®), colorectal tumor (cetuximab, Erbitux™) and several types of cancer (bevacizumab, Avastin™). In the case of an Rh-incompatible mother and child, there may be sensitization of an Rh- mother to the Rh antigen on the blood cells of the Rh+ child. This may put the remainder of the pregnancy, and any subsequent pregnancies at risk of fetal death due to hemolysis.
Immunoconjugates include antibodies linked to cancer-killing agents such as drugs, cytokines, toxins and radioisotopes. The results of the confocal microscopy imaging of the uptake of the same drug (Flutax-2) by this cell type is shown in Fig. The objective is for the antibody to act as a transporter for the cancer-killing agent, concentrating the agent directly in the cancer cell, with minimal damage to healthy cells.
Although conjugated antibodies showed toxicity in the past, more recent approaches under development appear to decrease unwanted side effects. For comparison, the following four different drug-delivery-system combinations were studied: (a) no drug, (b) free Flutax-2, (c) Flutax-2-GMO-MENs with no field. Anti-RhD antibodies are administered as part of a prenatal treatment regimen to prevent sensitization that may occur when a Rhesus-negative mother has a Rhesus-positive fetus. Pharmaceutical companies are developing immunoconjugates independently, forming partnerships with specialized players and even acquiring small biotech companies that are focused on the field of immunoconjugates.Although the challenge of their potential immunogenicity requires special attention, there are several practical advantages to immunoconjugates over single antibodies. We believe that this function was achieved due to the localized electroporation effects induced by the MENs in the vicinity of the cancer cell membranes when exposed to an external magnetic field. The advantages of immunoconjugates over single antibodies make them crucial players in new cancer therapy developments.
To refer to the effect at the nanoscale, we used the new terminology, “NANO-ELECTROPORATION.” The experiments were conducted to separate the two core field-dependent processes according to the main hypothesis. The specificity to the cancer cells was defined not just by the typical HER-2 antibody chemistry but also by the new physical mechanism that relied on the significant difference in the threshold electric field between the healthy and cancer cells. This situation has changed in recent years and mAbs are now the largest class of biological therapies under development, representing a multi-billion dollar worldwide market. This threshold field was measured to be of the order of 30?Oe and above 200?Oe for the SKOV-3 and HOMEC cells, respectively.
As reported recently by Scolnik [51], the 22 mAbs currently marketed in the US have a sales growth rate of 35% compared to less than 8% for small-molecule drugs. Moreover, these experiments indicated that this remote-magnetic-field-triggered electric-field-defined specificity to the cancer cells resulted in a more pronounced eradication of the cancerous cells. Oncology and autoimmune diseases are the most successful indications for these drugs, with five mAbs having sales in excess of $3B. The percentage of the cell-penetrated drug was increased by at least a factor of five compared to the traditional antibody-mediated targeting (figure 4).
Thanks to basic research, researchers are identifying new biomarkers, which could be potential targets for mAbs.
In addition, after the drug was efficiently transferred through the tumor cell membrane by the field-controlled MEN-initiated nano-electroporation, eradication of the majority of the cancer cells (without affecting the healthy cells) was observed within a 24-hour period of a low-energy 30-Oe treatment (figures 5 and 6).To achieve adequately high efficacy of the drug delivery, the value of the release field, Hr, was chosen to be higher than the value of the threshold field to penetrate through the membrane, Hth, for the cell of the same type.
They have a similar basic structure comprising of four polypeptide chains held together by disulfide bonds.
There are currently numerous mAbs at various developmental stages and it is expected that many of them will be available for clinical use in the near future.
It can be reminded that the release field is defined by the binding force between the MEN and the drug, while the penetration threshold field, Hth, is mostly determined by the electric properties of the cell membrane that lead to the localized electroporation effects. We could control the release field by the proper selection of the intermediate layer between the drug and the MEN. Nanoscale and Nanostructure-Based therapeuticsChemotherapy, radiation therapy and surgery are the most common types of cancer treatments available today. More recent treatments, which are at various stages of development, include angiogenesis inhibitor therapy, biological therapies (including interferons, interleukins, colony-stimulating factors, monoclonal antibodies, vaccines, gene therapy and nonspecific immunomodulating agents), bone marrow and peripheral blood stem cell transplantation, laser therapy, hyperthermia, photodynamic therapy and targeted cancer therapies [52].In the last two decades, a large number of nanoscale and nanostructure-based therapeutic and diagnostic agents have been developed, not only for cancer treatment but also for its prevention and diagnosis [53]. S1, by choosing different intermediate layers we could control the initial release field in a wide range, from less than 10?Oe to over 200?Oe.
Targeted cancer, hyperthermia, photodynamic and gene therapies are just some of the cancer treatments that use engineered nanomaterials.
These therapies can be used in isolation or in combination with other cancer treatments, thereby taking advantage of their ability to target tumors (actively or passively), to respond to physical or chemical stimulation (internal or external) and to deliver therapeutic genes to the cell nuclei.The main objective of nanomaterials in cancer treatment is to deliver a therapeutic moiety to tumor cells in a controlled manner (depending on the required pharmacokinetic) while minimizing side effects and preventing drug resistance. Nanoscale and nanostructured materials may also be used in diagnosis to detect and prevent pathologies as soon as possible, ideally being able to sense cancer cells and associated biomarkers. Examples of various nanostructured materials with potential applications in oncology are shown in Figure 3.The advantages of biocompatible nanomaterials have contributed to their significant expansion in cancer treatment. 2A, the spectrophotometry measurements of the absorbance at 230?nm (for PTX) indicated that only 1 minute of field treatment at a 66-Oe d.c. Targeted therapies for oncology are predicted to reach a 30 billion euro global market by 2015 [54]. The total market for nanobiotechnology products reached as high as $19.3 billion in 2010 [55]. Table 2 compiles some of the clinically approved nano-based therapeutics for cancer treatment and diagnosis. Many other nanoscale or nanostructure-based therapeutic and diagnostic agents are currently in clinical trials at various stages of development. This complex dependence can be explained by the fact that the external field effectively reduces the energy barrier that holds together the MEN and the drug while an increase of the treatment duration increases the temperature-induced probability to overcome the barrier or, in other words, break the bond.
As for the frequency dependence, in our previous paper we explained the underlying physics through field torque effects that break the bond as the frequency increases14. A year later, 50 ongoing clinical trials using nanoparticles for cancer were mentioned by Bergin [55], and at present, there are more than 70 clinical trials under development [56].
This large number of commercialnano-based therapeutics for use in cancer treatment is also reflected in the exponential increase in scientific publications and patents involving nanomaterials in recent years. Figure 4 shows the evolution over the last decade in the number of published scientific papers and issued patents involving nano-based applications developed to fight cancer. The number of papers and patents involving traditional forms of therapy (chemotherapy, radiation therapy and surgery) grew linearly over the last decade.
However, the use of the terms “nano-” and “cancer” has shown exponential growth over the past decade, demonstrating a major focus on nano-based tools applied to cancer treatment and diagnosis.
These fragments are then called Fab since they contained the antigen binding sites of the antibody.
Recent advances in the use of nanoscale and nanostructured-based therapeutic agents in cancer treatment are reported below.
The rapid cellular proliferation of these cells is also exploited by coupling the nanoparticles with different biological agents, such as folic acid. The rationale for coupling these carriers with folic acid is that the folate receptor is over-expressed in a broad range of tumor cell types, including solid and hematological malignancies [57].
Once it has reached the target, the cargo is released into the interior of the cell, and ideally, a signaling marker attached to the vector will aid the physician in visualizing the tumor.
S8), the MENs's field action didn't trigger any significant temperature changes in the field and frequency range under study. Such a vector may also be grafted with a moiety (usually PEG), which retards recognition by the reticulo-endothelial system (RES) to increase nanoparticle systemic circulation. In addition to recognition moieties, carried drugs and signaling elements attached to nanoparticles, numerous authors have also envisioned and designed vectors with additional functionalities, including cell-penetrating moieties, combinations of several drugs, combinations of drugs and genes, prodrugs (which become drugs upon biochemical modification by tumor cells), stimulus-sensitive agents that can be externally triggered and molecules for evaluating therapeutic efficacy. The more functionality added to the vector, the better the chances of reaching the target; however, its chances of being detected by the RES also increase. Therefore, currently marketed nanoparticles use passive targeting and active targeted nanoparticles are still being developed. Again, according to the main hypothesis, to ensure the specificity to the cancer cells, it is important to maintain the remote field above the release value for the tumor cells but lower than the threshold value for the healthy cells. For this reason, single-step synthesis of targeted nanoparticles by self-assembling pre-functionalized biomaterials provides a simple and scalable manufacturing strategy [59]. It has four polypeptide chains - two identical ''heavy chains'' and two identical ''light chains''. Mass production is also a serious concern and continuous synthesis procedures are therefore still being sought.
Specifically, the GMO-MENs field-treated HOMEC cells showed negligible drug intake per 1?mg of the cellular protein content. There are five types of mammalian Ig heavy chain denoted by the Greek letters: I±, I?, Iµ, I?, and I?. It can be noted that after a 24-hour 30-Oe field treatment by GMO-MENs, approximately 95 and 34% of HOMEC and SKOV-3 cells, respectively, remained viable (Fig. Immunological memory, created from the primary response to a specific nanoparticle, provides an enhanced response to secondary encounters with the same type of nanoparticle.
As an example, the recognition of PEGylated liposomes by anti-PEG antibodies has been reported to occur between 2 to 4 days after the first administration of PEG-liposomes, leading to fast clearance from circulation [54].
When the treatment was extended to 36 hours, the percentage of viable cells fell to approximately 85 and 10% for HOMEC and SKOV-3 cells, respectively. Finally, one of the last major barriers to achieving the transition of targeted nanoparticle use into clinical practice is the complete understanding of potential toxicological properties of these materials, along with their exact pharmacodynamics and pharmacokinetics.In spite of these hurdles, many research groups are focusing their efforts on solving them. Other groups are also directing their efforts towards designing more efficient targeted nanoparticles for cancer treatment in terms of structure, morphology, biocompatibility and surface functionalization. Some of those advances will be described later in this document.Novel targeted theragnostic nanoparticles have been synthesized and their bi-functionality demonstrated.
The nanoparticles were also able to function as tags for spectroscopic detection with surface-enhanced Raman spectroscopy. Magnetic targeting has also been used as a physical method for targeting and visualizing tumors. In this area are two heavy chains that contribute two or three constant domains depending on the class of the antibody. Effects of magnetic targeting on the extent and selectivity of nanoparticle accumulation in tumors of rats harboring orthotopic 9L-gliosarcomas were analyzed using magnetic resonance imaging (MRI) [62]. The particles size distribution was measured using dynamic light scattering method (Malvern-Zetasizer).Preparation of GMO-MENsIn-order to load the PTX drug onto the MENs' surface, the nanoparticles were first coated with GMO to adjust the release field at about 30?Oe as required for this application. The immunoglobulins present on the B-lymphocyte surface send in signals to the cytoplasmic and nuclear electors. These also deliver the antigen to the cell where it can be destroyed, processed and returned to the cell surface to be presented by MHC class II molecules to antigen-specii¬?c T helper cells. The mixture was then incubated for 12 hours while being slowly rotated in order to achieve uniform coating. In this targeted delivery system, anti-HER2 single chain Fv fragments were attached to the end of PEG chains located on the surface of liposomes.Targeting via extracellular activation of the nanocarrier is a promising method for achieving active targeting using physiological stimuli present in the tumor environment. Upon completion of the incubation process, the nanoparticles were centrifuged at 20,000?rpm for 20 minutes at 10°C.
Triggering mechanisms that only release the transported cargo of nanocarriers into the tumor environment take advantage of its acidic pH and uncontrolled enzyme production.
A complete description of these systems is reported elsewhere [58].Tumor targeting of prodrugs that become active once they reach tumor cells is another novel strategy for avoiding unwanted side effects of the drug, and it allows for the delivery of large doses of drugs.
The humoral immune system helps in destroying external pathogens and prevents spread of intracellular infections. The results of the energy dispersion spectrometry (EDS) that depict the materials composition of GMO-MENs are summarized in Fig. S10.Preparation of HER-2 biomarker antibody conjugated GMO-MENsHER-2 biomarker antibodies were covalently attached onto the GMO-MENs' surface according to the protocol described by Kocbek et al34.
In-order to covalently attach the HER-2 antibodies, the nanoparticle surface was preliminarily functionalized.
Then, the sample was centrifuged at 14,000?rpm for 10 minutes at 10°C and the pellet was washed three times with 1?ml of the PBS buffer (pH 7.4).
The immune system generates large number of antibodies that can recognize virtually all possible antigens present in pathogens and their products. These may be on invading microbes such as bacteria, viruses, and parasites as well as environmental antigens. The solution was incubated for 2 hours while being rotated slowly and kept further at 4°C overnight. Antibodies can be produced against all types of molecules including carbohydrates, nucleic acids and phospholipids but are best suited to bind against a protein.
The pellet was washed thrice with 1?ml of the PBS buffer (pH 7.4) to remove any excess antibody. The supernatant was collected to determine the amount of the unconjugated HER-2 protein by comparing to the standard plot. The binding prevents the access of the pathogen into the cells and prevents infection or destruction of host cells.
The percentage of the conjugated HER-2 was obtained using the following expression: the percentage of HER-2 conjugated = (the total amount of HER-2 added – the amount of the unconjugated HER2 present in the supernatant) ? 100.
Antibodies also block the binding of the bacteria to host cells by binding to cell-surface proteins. Then, the solution was centrifuged at 14,000?rpm for 10 minutes at 10°C to remove any unbounded drug. The supernatant was isolated and absorbance was measured spectrophometrically at 230?nm using Cary-100 UV-VIS spectrophotometer30. A standard calibration plot for PTX was obtained by varying the drug concentration from 5 to 80??g in 1?ml of the MPBS solution and the absorbance was measured at 230?nm. The absorption maxima and the standard linear calibration plot of PTX at different drug concentration values are shown in Fig. Human Ovarian Microvasicular endothelial cells (HOMEC) from ScienceCell (Carisbad, CA) and were cultured in endothelial cell medium with endothelial cell growth supplement (1%), fetal bovine serum (5%), and penicillin-streptomycin (1%).
All the cells were cultured at 37°C cell incubator with a 5% CO2 and humidified atmosphere.Fluorescence measurements and confocal imaging of drug uptake by SKOV-3 cells for different drug-carrier combinationsCellular drug uptake measurements and fluorescence imaging were performed using an Oregon Green® 488 paclitaxel (also called Flutax-2). IgG can thus neutralize foreign antigens and protect epithelial cells from infectious agents acting as first line of defence.
For the fluorescence measurements, the SKOV-3 cells were cultured in 24-well plate at a density of 2 ? 105?cell per well. These include phagocytic cells like macrophages and neutrophils, T cells like natural killer cells, and eosinophils and mast cells. In addition, a set of controls containing no drug for all the combinations was cultured under similar conditions. The cell culture plates containing the Flutax-2-MNs and Flutax-2-GMO-MENs were kept under a 30-Oe field. Upon completion of the 10-hour incubation process, the cells were removed from incubator and the cell culture medium was discarded. Complements are series of plasma proteins that help in release of chemical mediators from mast cells (mast cell degranulation), phagocytosis (eating up of bacterial and microbial cells by macrophages) and cell lysis (breaking down or bursting of the invading cells).
Then, 1?ml of dimethyl sufloxide (DMSO) was added to each well and incubated for 2 hours at 37°C.
Complement activation begins when the C1q molecule binds to antibody molecules attached to the surface of a pathogen and triggers the classical pathway of complement activation. After two hours, a rubber policeman was used to ensure the complete removal of the attached cells. The main functions of the complements are to enable phagocytes to destroy bacteria that they would otherwise not recognize. The solution was centrifuged at 14,000?rpm for 10 minutes at 4°C to obtain the cell lysate.
The cell lysate along with the in-taken Flutax-2-GMO-MENs was collected and measured for the fluorescence of Flutax-2 (using BioTek instruments, synergy HT) at ?ex = 496?nm and ?em = 524?nm to determine the concentration. All the fluorescence measurements were recorded by subtracting the corresponding controls to adjust the background fluorescence from the cellular components. The protein content of the cell lysate was determined using Bio-Rad protein assay kit (Braford method) by measuring the absorbance at 595?nm using a Cary 100 UV-VIS spectrophotometer.As for the imaging studies, the cells were cultured on glass cover slips (1 ? 1 in2) pre-coated with the poly-L-Lysine (used as a cell adhesion promoter) in a 6-well cell culture plate at a density of 5 ? 104 were cultured and let rested for about 10 minutes. The antibodies coat the surface of the pathogen and allow binding of their Fc domains to Fc receptors present on elector cells.
The macrophages and neutrophils then engulf the pathogen and internalize the microbe causing its destruction. With one edge they protect the body from microbes and with the other they can cause severe allergic reactions to relatively harmless proteins and other molecules present in food, environment, medicines etc. After the incubation process, cover slips were washed three times with the PBS buffer and fixed with 4% paraformaldehyde for 30 minutes followed by washing thrice with the PBS buffer. The cover slips were mounted onto a glass slide using a mounting medium (ProLong Gold Antifade Reagent). When it binds to multivalent antigens there is activation of the mast cell, which releases chemical mediators stored in granules and capable of mediating local ini¬‚ammatory reactions.
The excess mounting medium was removed by placing a small piece of Whatman paper around the edges.
After the samples were dried for 2 hours, they were imaged through confocal microscopy (TCS SP2, Leica Microsystems, Germany) at 488?nm (100%) illusion of an argon-ion laser using 60? oil immersion objectives with a high numeric aperture and 1? confocal electronic zoom settings to visualize cells. Since each of the antibodies are different for each of the antigens, the body needs to be capable for generating these proteins. To create the variety of antibodies thus the body adopts complex mechanisms from the relatively small number of antibody genes. This means generation of distinctly different antibodies due to different variable domains on the antibodies. The variable region of each immunoglobulin heavy or light chain is encoded in several pieces on the genes.
In this there is usually no alteration in the L chain or in the variable portion of the H chain and thus there is no change in antigen-binding specificity.
The genes coding for the antibodies in these B cells undergo high rate of point mutation or point changes in genetic codes for antibodies. While some of the generated antibodies are weak, some have a stronger affinity for the antigen. Those B cells that produce these strong antibodies as a result of the mutations are preserved and proliferated while the others die off.
This they did by transferring serum produced from animals immunized against diphtheria to animals suffering from it.
He speculated that this interaction induces the cell exhibiting the receptor to multiply and produce more copies of the same receptor.
The biomolecule responsible for these actions was termed antitoxin, precipitin and agglutinin. Kabat in 1938 had also shown heterogeneity of antibodies through ultracentrifugation studies of horses' sera.
James Gowans in 1959 showed that lymphocytes had a role in mediating both cell-mediated and humoral responses.



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