Dual-targeted Nanoparticles for the Combination Therapy of Glioblastoma Multiforme

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Dual-targeted Nanoparticles for the Combination Therapy of Glioblastoma Multiforme

Primary Aim:To achieve dual-targeted nanoparticle approach and overcome the resistance for temozolomide therapy for the cure of grade-IV glioblastoma multiforme.

Objective:

Abstract:

Glioblastoma multiforme (GBM) is one of the most aggressive forms of cancers in the United States. Due to its increasing mortality rate and a 5-year survival of less than 35%, makes it necessary to design a targeted drug therapy. Temozolomide (TMZ) and radiation therapy has been the standard therapy used for the treatment of GBM patients which extends the survival time for the patient up to fifteen months. However, there has been an increase in cases for TMZ resistance seen in the cancer patients. Hence, the need for a novel targeted therapy is required to maintain the efficacy with prolonged treatment. Here, I suggest a novel targeted therapy using a prodrug approach of TMZ along with natural products like curcumin and 20S-protopanaxadiol (PPD) as a therapeutic agent in a dual-targeted nanoparticle-based drug delivery. There has been a lot of studies which have reported about the resistance in the current therapy of TMZ. Hence, it becomes a rationale to design and formulate a novel targeted approach to GBM. The blood-brain barrier (BBB) being one of the most difficult hurdles to overcome for achieving the desired tumor concentration of the drug in GBM. The prodrug form of TMZ will be delivered along with the natural product molecules inside the cancer cell to show the maximum therapeutic action against GBM. At a different ratio of nucleolin targeting (F3) peptide: tLYP1 targeting peptides: EGFRvariantIII antibody (scFv fragment) will be used to decorate the nanoparticle (NP) to achieve the maximum therapeutic action against GBM. The in-vitro and in-vivo studies (dual-targeted TMZ prodrug loaded NP, TMZ prodrug loaded NP, blank NP and only TMZ loaded NP) will be carried out using xenograft model developed using Wistar rat model.

Development of dual-targeted nanoparticles (NP) based combination therapy by using temozolomide (TMZ) prodrug(1) (curcumin conjugated) along with the anti-EGFRvariantIII (anti-EGFRviii) scFv fragment(2, 3) used for the treatment of glioblastoma multiforme (GBM). Maximum therapeutic efficiency will be achieved by overcoming the resistance for TMZ by cancer cells and inducing cytotoxicity by achieving high drug loading inside the nuclei of the cancer cells. Also, inhibiting the overexpressed EGFRviii(4) in the cancer cells to prevent metastasis and achieve complete cure of grade-IV GBM in adult patients.

Introduction:

Cancer is a disease condition in which the normal cells begin to multiply and spread abnormally in any part of the body. Glioblastoma multiforme (GBM) is one of the aggressive types of the brain tumor that forms from the glial tissue (supportive) of the central nervous system (CNS) which includes both brain, and spinal cord. It is also called grade IV astrocytoma. CNS consists of two types of supportive cells: astrocytes and oligodendrocytes. Most of the gliomas are associated with either astrocytes or oligodendrocytes or a mixture of both cells. Gliomas are graded as per the tumor resemblance to healthy brain cells, which is then graded from I – IV based on their degree of aggressiveness. A grade I glioma is usually a benign, while the tumor that grades from II to IV are tumor with an increasing level of aggressiveness and malignancy potential(5).

The blood-brain barrier (BBB) being one of the major obstacles to the drug delivery in the CNS. Even though the primary function of the BBB is to keep unwanted substances away from the brain, the BBB acts as an obstacle for many drugs that have potential therapeutic applications in neurodegenerative disorders. The BBB prevents the entry of most molecules, except ones that are small and lipophilic. The studies have reported that they compose of tight junctions between brain capillary and endothelial cells. In addition to endothelial cells, pericytes, astrocyte end-feet, microglia, and a basement membrane make up the BBB(6). The BBB being one of the most stringent obstacles to drug delivery, mainly due to its anatomy. The endothelial cells in the BBB are attached to each other by tight junction protein complexes, such as claudins, occludins, and junctional adhesion molecules (JAMs) which indicate that the junctions at the BBB are very tight resulting in more restrictions for the paracellular transport of materials across BBB(7). Another obstacle that exists for drugs in the BBB as a transcellular barrier is the presence of drug transport systems, which are also known as ABC-transporters (ATP-binding cassette transporters). These actively pump out substances that have reached the endothelial cell membranes back into the blood stream. In addition to this function, ABC-transporters play a critical role in pathology. For instance, cancer stem cells have increased levels of ABC transporters which allow them to have increased resistance to chemotherapy and cancer drugs(8). It is crucial to note that greater than 98% of small molecule and approximately 100% of large molecule drugs cannot cross the BBB(9). Researchers implement various techniques to surpass the BBB, including the osmotic opening of BBB, the use of prodrugs, liposomes(10) and nanoparticles(11).

Gliomas are not a specific kind of brain cancer. Glioma is a term used for a group of tumors that originate from glial cells. Several tumors can be considered gliomas, including glioblastoma (also known as glioblastoma multiforme), astrocytoma’s, oligodendrogliomas, and ependymomas. Most fast-growing brain tumors are gliomas. Other types of brain cancers are mixed gliomas, meningiomas, medulloblastomas, gangliogliomas, schwannomas and craniopharyngiomas. There are few types of tumors which can develop into brain cancer like, chordomas, non-Hodgkin lymphoma, and pituitary tumors(12).

The current statistics depicts that brain cancer is one of the fatal aggressive cancers in the United States. Recently it has been ranked tenth leading cause of deaths in cancer patients in the United States, as per the reports of 2017 by American Cancer Society (ACS). It is estimated about 23,800 new cases will be diagnosed in 2017 including 13,459 men and 10,350 women. Also, 16,700 people will die because of brain cancer including 9,620 men and 7,080 women. Overall, brain cancer signifies 1.4% of all the new cases in the US(13).

Despite the increase in the cancer research, there has been no meaningful change in past 50 years in the cure of brain cancer. There have been some new targets discovered for the treatment of brain cancer, like, overexpression of EGFRviii(14), PD-L1(15, 16), MDA-9/Syntenin(17), etc. these targets have been few of the important targets for the cure of brain cancer and studies are carried using targeted drug-delivery systems.

Hypothesis and Specific Aim:

Hypothesis 1: The resistance seen in the treatment of TMZ followed by the increase in the metastasis and relapse of the GBM followed by short duration of survival of the patients suffering from GBM(18). Therefore, its gives a rationale to design a novel targeted drug delivery approach for the treatment of GBM. Furthermore, I will like to propose a combination therapy using prodrug form of TMZ along with curcumin(19) and 3, 12-diacetylated protopanaxadiol (PPD)(20) conjugated with PEG-PLA, followed by matrix metalloproteases-2 (MMP-2) mediated scFv-EGFRviii antibody therapy(21). Moreover, nucleolin and tLYP1 peptides will be used for the dual-targeted PEG-PLA based nanoparticle drug delivery(22). It seems to be a highly potent and novel targeted approach for the treatment of GBM. Since nucleolin is highly expressed on the endothelial cells of the BBB along with the cell membrane of cancer cells in GBM. Hence, it will help to increase the uptake of NP from the blood and direct the drug to reach into the nucleus. The size of the NP is expected to be <200nm, hence this will also help in the diffusion of the nanoparticles from the BBB. EGFRviii is significantly overexpressed only in cancer cells responsible for GBM and leads to resistance to TMZ therapy. Hence, it becomes an important target for the treatment of GBM. Therefore, it can be an efficient approach for the targeted prodrug delivery directed to the nucleus of the cancer cell.

Specific Aim 1: Preparation and evaluation of nanoparticles (NP) as a target specific drug delivery system for the cure of GBM. Nanoparticle will be synthesized using polyethylene glycol (PEG) and polylactic acid (PLA) using different monomer size of Mal-PEG for conjugating with the targeting ligands or antibody (scFv fragment). Target specific ligands will be attached to the polymers used, which will be synthesized before making NP. Mal-PEG-PLA will be used to attach the ligands to the surface of the NP. Also, PLA will be conjugated with protopanaxadiol (PPD) to incorporated it into the hydrophobic core. Simultaneously the Mal-PEG-MMP2 sensitive peptide-PLA will be synthesized and conjugated with the scFv fragment of anti-EGFRviii.

Hypothesis 2: TMZ resistance in GBM can be taken care of by following ways, first is an increase in the concentration of TMZ inside the tumor cells targeted drug-delivery approach, which is the nucleus of cancer cells. The MMP-2 based anti-EGFRviii therapy using a scFv fragment to inhibit the overexpressed proteins which help to overcome the resistance in TMZ drug regimen. Inhibition of the metastasis in the tumor cells will be achieved by using natural products like curcumin and PPD loaded in the same NP.

Specific Aim 2: Cellular uptake and antibody fragment and peptide binding assay will be carried out. F3 peptide (CKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK) will be used for targeting nucleolin receptors, and tLYP1 peptide (CGNKRTR) will be used to synergies the uptake of NPs in the cancer cells(22). The labeled-antibody conjugated to the NP will be targeted to the cancer cell lines and will be evaluated for binding studies by flow cytometric quantification technique. Similar binding studies will be carried out for F3 and tLYP1 peptide-based nucleolin-targeted delivery using multiple cancer cell lines for GBM.

Hypothesis 3: Desiring it to be a highly potent and target specific approach. The in-vitro studies will be carried out using multiple cell lines, expecting it to successfully transport through BBB and the plasma membrane of the tumor cells. The significant concentration will be seen inside the nucleus followed by blocking of EGFRviii to overcome the TMZ resistance. Anti-EGFRviii will significantly bind to the overexpressed EGFRviii which will be seen through the confocal images using fluorescent dye-tagged antibody. There will be a significant reduction of protein expression levels of EGFRviii that will be observed using western blot technique. Optimized NP formulation will be utilized for further characterization studies.

Specific Aim 3: The specific cell lines will be used for the transport, uptake in-vitro studies of dual-targeted drug loaded NP. Characterization of final nanoformulations will be done using dynamic light scattering (DLS) machine for size and zeta-potential, the osmolarity, pH, stability and dilution studies will be carried out for final NP formulation. Finally, different brain cancer cell lines will be used for cytotoxicity studies using prodrug loaded dual targeted NP; prodrug loaded NP, blank NP, and free TMZ/curcumin/protopanaxatriol.  Hence, the MTT and LDH studies will be carried out using specific cell lines.

Hypothesis 4:Thematrix metalloproteases (MMP2) enzyme is present at higher concentration in extracellular matrix around the cancer cells also seen for GBM. MMP2 can be used as a mediator for the treatment of GBM by cleaving the MMP2 sensitive peptide conjugated antibody from the surface of NP. Thus, the scFv fragment will be directed towards EGFRviii which is significantly overexpressed in cancer cells. Hence, the dual-targeted therapy leads to inhibition of growth of cancer cells along with inhibition of metastasis, achieved by overcoming the resistance for TMZ in cancer cells of GBM.

Specific Aims 4: The studies will be carried out to see the effect and duration of release of antibody conjugated with the polymer on the surface of NP. Hence, time-based studies will be conducted; the inhibition will be measured looking at the protein concentration of EGFRviii in the cancer cells at different time points.

Hypothesis 5: Different brain cancer cell lines will be selected and grown in the respective growth medium of the given cancer cell lines, followed by administration of prodrug loaded dual-targeted TMZ prodrug NP, TMZ prodrug NP, blank NP, and free TMZ will be used in the study. The in-vivo animal studies will be done to verify the effect of the target based approach.

Specific Aim 5: In-vivo tumor internalization, biodistribution of targeted NP and toxicity studies will be carried out in xenografts rat models.

Background and Significance:

Glioblastoma multiforme being one of the aggressive cancers in the United States and worldwide. With the increasing mortality rate, gives us a rationale to design a target based drug-delivery system which helps in the treatment of GBM. In the normal glial cells which usually expresses nucleolin receptors only on the nuclear membrane, which is responsible for the delivery of the molecules from the cytoplasm to the nucleus(22), this receptor is overexpressed in the cancer cells of GBM. It is expressed on the endothelial cells of BBB and the plasma membrane of the cancer cells responsible for GBM(23). Thus gives me a rationale to use it for targeting the drug loaded NP. The increase in uptake was seen inside the cancer cell through nucleolin with the help of F3 peptide (CKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK) along with the tLYP1 peptide (CGNKRTR), confirmed by in-vivo studies in the previous works(22). Hence both the targeting peptides (F3 and tLYP1) are conjugated with Mal-PEG-PLA-PPD to achieve the desired therapeutic concentration of TMZ prodrug in the nucleus of the cancer cells. Currently, TMZ is being used along with radiation therapy for the treatment of GBM(18). TMZ being a DNA-alkylating agent and used as a first-line drug for the treatment of GBM; it has been seen to be less effective due to the resistance developed by the tumor cells. First, the prodrug approach of TMZ conjugated with curcumin will be used for the treatment of GBM. Curcumin being a natural product, it shows multiple pathways for inhibiting the cancer cell growth by disrupting molecular signaling and inducing apoptopic pathway and also reduces invasion and metastasis in cancer cells. Curcumin when administered with the existing chemotherapeutics, it enhances its effect(19, 24). Therefore, prodrug approach helps in achieving the therapeutic concentration at the target site inside the cancer cells of GBM. Second, the anti-EGFRviii scFv fragment is used for overcoming the drug resistance of TMZ by inhibiting EGFRviii and inducing apoptosis in the cancer cells causing GBM. The antibody will be released as soon as it reaches near the cancer cell with the help of matrix associated metalloproteases (MMP) enzymes present abundantly. The MMP sensitive peptide (Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln) will be cleaved from the surface of NP which in turn will let the antibody fragment bind with overexpressed protein (EGFRviii) and lead to inhibition to the growth of cancer cells. Further, the receptor-mediated endocytosis will be followed by endosomal escape with the help of basic amino acids present in F3 and tLYP-1 peptide on the surface of NP. Moreover, with the help of F3 peptide, the prodrug will be delivered it inside the nucleus of cancer cells. The TMZ prodrug will be converted to an active form with the aid of esterase present in the cell, which will lead to alkylation of DNA followed by inhibitions in DNA replication leading to cell apoptosis. Finally, the protopanaxadiol (PPD) and curcumin will also be released by esterases to it active form to show its therapeutic effect. The diacetylated for of PPD shows to have a significant antiproliferative effect in cancer cells(20). Thus, such a combination effect will have a cumulative and synergistic effect which will also be useful in reaching the cancer stem cells of GBM. It will also inhibit the metastasis and relapse of cancer which is the major hurdle in the treatment of GBM. With the help of strong chemistry and desired formulation, we may be able to cure grade-IV GBM in adult patients. Using the basic concepts of molecular mechanisms of cancer metastasis, drug development, formulation and general chemistry may yield a targeted cancer therapy with the enormous potential for clinical success. Here, I have created a novel strategy to inhibit progression of cancer and cause apoptosis in GBM synergistically.

Fig 1: A schematic diagram of dual-target NP loaded TMZ prodrug and its nanostructured core.

Research Design and Methods:

  1. Prodrug Synthesis(1, 25):

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

R = Curcumin

  1. Preparation of Mal-PEG-PLA-PPD:

Firstly, Mal-PEG-PLA-COOH and 3, 12- diacetylated protopanaxadiol will be conjugated using 1:1 ratio (w/w) through esterification. The polymer will be purified by chromatographic techniques and purity will be analyzed by HPLC.

  1. Preparation and evaluation of scFv-EGFRviii antibody, F3 and a tLYP1 peptide conjugated with NP for targeted delivery:

C.1. Synthesis of Mal-PEG-peptide-PLA:

The Mal-PEG-NHS will be coupled with the N-terminal NH2 of the first glycine, is part of the MMP2 sensitive cleavable peptide (NH2-GPLGIAGQ-COOH peptide), will be mixed and stirred in the carbonate buffer (pH 8.5) at 4°C overnight. The excess of peptides will be removed by dialysis against distilled water. Mal-PEG-peptide was identified by RP-HPLC on a reverse phase C-18 column HPLC system. The chromatograms will be collected at 214 nm using gradient elution method. Followed by conjugation of C-terminal COOH group of terminal glutamines with PLA. The reaction will be monitored using TLC followed by staining using the Molybdenum Blue Spray Reagent. Finally, the synthesized polymer will be purified using gel permeation chromatography (GPC) and will be further used for conjugation of antibody on the surface of NP(21).

C.2. Synthesis of Ab-PEG-peptide-PLA:

Mal-PEG-peptide-PLA will be used to attach the thiol terminal of a modified scFv fragment of the antibody (scFv-EGFRviii) to conjugate on the surface of NP for targeted delivery and inhibit of EGFRviii action. The scFv fragment will be modified by attaching two Cys residues on the terminal NH2 to act as the linker between scFv and the NP. The purification will be done using ultracentrifugation, followed by purity analysis will be confirmed by using gel permeation chromatography and analyzed using UV-detector. The antibody (scFv EGFRviii) conjugation on the NP will be confirmed using a fluorescence moiety(26).

C.3. Evaluation of Ab-PEG-peptide-PLA conjugation:

The immunological activity of the antibody on the multifunctional NP will be determined by ELISA. It will be checked that antibody and antibody-modified MMP2-responsive NP have a similarly high binding affinity with overexpressed proteins, while negative controls, IgG and unmodified NP will show no binding. The high concentration of antibody could saturate the plate which will not be possible in its real binding affinity. Therefore, linear range concentrations of Ab will be selected to compare the binding affinities of the NP formulations. After the treatment with MMP2, Ab-PEG-Gly-Pro-Leu-Gly will be liberated from the nanoparticle due to the cleavage of the MMP2-cleavable linker between PEG and PLA. The liberated Ab-PEG-Gly-Pro-Leu-Gly will stay conjugated, which will show a binding affinity as similar the non-treated nanoparticles. However, the binding affinity of the MMP2-responsive dual-targeted nanoparticle will significantly decrease after the liberated Ab-PEG-Gly-Pro-Leu-Gly will be removed using dialysis bags (MWCO 300,000 Da).  The ELISA results will suggest that scFv anti-EGFRviii fragment is successfully conjugated to MAL-PEG-peptide-PLA on the surface of NPs via the MMP2-cleavable linker and did not lose its immunological activity(21).

  1. Conjugation of PLA-PEG-Mal with F3 and tLYP1 peptide through sulfide bond linkage:

D.1. Synthesis of F3 and tLYP1-PEG-PLA:

Mal-PEG-PLA-PPD will be used to attach the modified terminal of F3 and tLYP1 peptide with cysteine for conjugation with the surface of NP(22). Michael additive reaction between maleimide and the thiol group will be performed to form the C-S bond linkage so as to achieve target based delivery using nucleolin as a target protein which detects F3 sequence and helps in the receptor-mediated endocytosis of NP from BBB and cell membrane of cancer cells.

The conventional NP usually have low bioavailability and are easily absorbed by the RES. Therefore, NP’s have been used, and various techniques have been developed to overcome this problem by coating the surface of the NP with hydrophilic polymers, such as polyethylene glycol (PEG). PEG possesses high flex­ibility, desired hydrophilicity, anti-phagocytosis against macrophages, resistance to immunological identification (as antigen), prevent protein binding, and increases biocompatibility, which enables the extensive application in developing the PEGylated NP for delivering various drugs.

D.2. Evaluation of F3/tLYP1-PEG-PLA conjugation:

The synthesized NP-F3/tLYP peptide conjugate will be identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. To determine that specific nucleolin uptake through F3 targeted ligand NP by the glioma cells and will be quantified by flow cytometry.

  1. Preparation of dual-targeted NP loaded with TMZ prodrug:

The nanoparticles (NP) loaded with TMZ prodrug will be prepared through the emulsion/ solvent evaporation technique according to the procedure described [22]. The Ab-PEG-PLA-PPD, F3peptide-PEG-PLA-PPD, and tLYP1peptide-PEG-PLA-PPD will then be dissolved different proportions in dichloromethane, followed by the addition of 1% sodium cholate aqueous solution, emulsified by sonication (280 w, 30 s) with Probe Sonicator in ice water bath. Then the emulsion will be added into 0.5% sodium cholate aqueous solution under rapid magnetic stirring for 5 mins. The emulsion was subsequently applied to a rotary evaporator to remove the dichloromethane and concentrated by centrifugation at 20000 g for 45 min using a centrifuge. After discarding the supernatant, the nanoparticles were resuspended in distilled water and then subjected to a 1.5×20 cm Sepharose CL-4B column and eluted with 0.01 M HEPES buffer (pH 7.0) to remove the un-encapsulated TMZ prodrug(22).

  1. Characterization of Dual-targeted Nanoparticle:

F.1. Particle size, zeta-potential and TMZ prodrug loading/encapsulation efficacy of nanoparticle:

The particle size and zeta-potential of the dual-targeted NP’s will be characterized by photon correlation spectroscopy (Malvern Instrument) and potential nanoparticle analysis (ZET-3000HS, Malvern Instrument), respectively. The morphological examination of NP’s was performed via transmission electron microscopy (TEM) Besides; the NP’s were analyzed with Multimode Atomic force microscopy (AFM)(27). Loading efficiency (LE) and encapsulation efficiency (EE) will be determined drug-loaded nanoparticle with the help of high-performance liquid chromatography(22).

EE (%) = TMZ prodrug in the nanoparticle   x 100     LE (%) = Amount of TMZ prodrug in nanoparticle   x 100

Total amount of TMZ prodrug in dispersion                                     Nanoparticle weight

In-vitro release of prodrug TMZ from NP was investigated in PBS (pH 7.4) containing 0.5% Tween 80. One milliliter of NP solution was mixed with the same volume of PBS (pH 7.4) containing 0.5% Tween 80, sealed into dialysis tubes (MWCO-100,000 – 300,000), and incubated in 10 mL of PBS at 37oC for 7 days in the thermal bath on continuous stirring. At the predetermined time points, aliquots in the dialysis medium will be withdrawn, and HPLC will measure prodrug TMZ concentration. The cumulative release percentage (%)will be indicated by dividing the cumulative amount of TMZ prodrug recovered in the dialysis medium with the total amount in the NP solution(27).

F.2. In-vitro cell culture studies:

C6 glioma cells and bEnd3 cells will be cultured in F-12K medium and DMEM medium supplemented with 10% FBS respectively, 100 IU/ml penicillin, and 100 mg/mL streptomycin sulfate. Cell lines will be cultured in incubators maintained at 37oC with 5% CO2 under fully humidified conditions. All experiments will be performed on cells in the logarithmic growth phase.

Uptake studies of dual-targeted NP loaded with TMZ prodrug and effect of dual-targeted NP modified by F3 and tLYP-1 peptide on P-gp function and expression in bEnd3, and C6 cells will be studied. Followed by the intracellular distribution of dual-targeting NP will be carried out to quantify the amount of TMZ prodrug reaching the nucleus with the help of targeted approach. BBB model in vitro will be built, and transport studies across BBB model will be carried out in-vitro. In-vitro cytotoxicity studies will be carried out using MTT and LDH assay using C6 glioma cells and bEnd3 cells. Further quantification will be done using spectrophotometer and microplate reader(27).

F.3. In-vivo animal studies:

F.3.1. Establishment of in-situ glioma model (xenograft) for in-vivo studies:

Wistar rats (weighing 180-220 g) will be housed under standard conditions with free access to food and water. The in-situ glioma model will be established according to the reported method.

F.3.2. In-vivo distribution of dual-targeting NP in glioma:

Rats bearing intracranial C6 glioma will be formed as described above. Fourteen days after implantation, targeted NP will be injected into the tail vein of rats at a dose. At 2 h postinjection, the rats will be anesthetized with and treated with reported protocols. Then the brains will be removed, fixed for 24 h. Moreover, the brains were embedded in OCT and frozen at -80oC, sectioned at 10 mm. Following counterstaining with DAPI for 10 mins and rinsing with PBS, slides were visualized under fluorescence microscope.

F.3.3. In-vivoantitumor study:

The anti-tumor efficacy will be studied using C6 cells bearing in-situ tumor model. After tumor inoculation, the rats will be randomly divided into five groups (9 rats per group). Animals in the blank control group will be administrated with saline, and another group will be administrated with TMZ prodrug solution, TMZ prodrug NP and dual-targeted TMZ prodrug NP. Rats will be administered via tail vein at a dosage. Body weight at day 8, day 11, day 14 and day 17 post-glioma cells planted will be measured. Magnetic resonance imaging (MRI) will be helpful in the experiments to measure tumor volume(27).

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