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The broad long term objective of this new grant application is to develop novel melanotransferrin conjugated nanogels for brain tumors. Brain tumor continues to be a significant health issue in the USA and world-wide. The median survival for patients with GBM has not improved substantially over the past 30 years because the nonspecific and non-targeted nature of most of the drugs currently used and their inadequate delivery across the blood brain barrier and tumor cells. Advances in understanding of the cell biology of the BBB have opened new gates for improved drug delivery to the CNS. In past few years several carrier or transport systems and receptors that modulate the permeation of molecules have been identified in the BBB endothelium. Nanogels are very promising in drug delivery applications due to their high loading capacity that is unique for pharmaceutical nanocarriers. They have several advantages over the conventional polymeric nanoparticles such as high drug loading capacity; control of burst release, organic solvent is not required in the synthesis process and no energy input is required such as high-pressure homogenization and sonication. Chemotherapeutic agents can be encapsulate inside the polymeric matrix of nanogels to evade efflux transporter present on BBB and tumor cells; to achieve target specificity this nanogels can be functionalized with specific ligand (e.g. melanotransferrine). So our objective is to encapsulate the chemotherapeutic drug (Docetaxel) in nanogel and to functionalize this nanogel with melanotransferrine which is specifically endocytosed through low-density lipoprotein receptor-related protein (LRP) receptors which are highly expressed on BBB as well as human glioblastoma cells. This polymeric nanogels matrix degrades, releases the loaded drug inside the tumor cells, bypassing limitations imposed by the BBB and minimizing systemic exposure of the drug.
Therefore the Specific Aims of this new grant application are:
1. To prepare and characterize unmodified and surface modified docetaxal loaded nanogels
2. To study the uptake and cell proliferation of both surface modified and unmodified nanogel on rat brain microvessel endothelial cells (RBMEC)
3. To evaluate in vivo time and dose dependent concentration of docetaxal in the brain, following intravenous administrations of the nanoparticles, using brain microdialysis technique in an unconscious Wistar rat model
4. To evaluate in vivo time and dose dependent tumor size reduction, in Wistar rats with brain-implanted C6-glioma, with nanogels
Background and significance
Brain tumor continues to be a significant health issue in the USA and world-wide. Overall, they constitute some of the most malignant tumors known to affect humans and are generally refractory to all modalities of treatment. It is estimated that about 30,000 to 35,000 new cases of primary brain tumors will be diagnosed in the upcoming year in the USA (1-2% of newly diagnosed cancers overall) (1). Majority of brain tumors are malignant gliomas, often glioblastoma multiforme (GBM) and anaplastic astrocytoma (AA). Recent epidemiological studies suggest that incidence rates of primary brain tumors may be increasing for children and patients aged 70 years or older. The median survival for patients with GBM has not improved substantially over the past 30 years and is approximately 12-14 months. These survival data suggest that the current treatment of brain tumor patients is inadequate and unable to overcome the malignant biology of these neoplasms. Explanation for the poor results of chemotherapy includes the nonspecific and non-targeted nature of most of the drugs currently used and their inadequate delivery across the blood brain barrier and tumor cells (2).
Blood brain barrier
Blood brain barrier (BBB) is very specialized barrier system of endothelial cells involved in regulation of molecules accessing the brain and preserving brain homeostasis. The brain endothelial cells are sealed together by tight junction, having no fenestrations and very few pinocytosis vesicles. The system is highly protected from various toxins. Multiple drug efflux pumps are expressed on brain capillary endothelial cells as well as cancer cells. The most notable of theses is P-gp and also other members of multidrug resistance family are expressed either on luminal or brain side of the BBB. Advances in understanding of the cell biology of the BBB have opened new gates for improved drug delivery to the CNS. In past few years several carrier or transport systems and receptors that modulate the permeation of molecules have been identified in the BBB endothelium (3, 4).
Our drug of choice is Docetaxel (TaxotereÂ®), a novel, hemisynthetic, anti-cancer agent, structurally related to paclitaxel (Taxol) has shown efficacy in clinical trials against a variety of human tumors. Recently its antitumor activity against malignant gliomas has been checked. It is far more potent than many other anti-glioma agent including paclitaxel (5). Its mechanism of action is through inhibition of tubulin depolymerization resulting in microtubule aggregation and cell death (6). Historically, malignant glioma has remained refractory to conventional systemic treatment with decetaxel against tumors because it does not cross the BBB and actively efflux out by P-gp. To avoid systemic toxicity associated with intravenous administration (7, 8) and to achieve high concentration inside the brain, docetaxel can be encapsulated inside the polymeric material (9, 10).
We have selected nanogels as carrier, which are crosslinked polymeric particles, which can be considered as hydrogels if they composed of water soluble/swellable polymer chains (e.g N-isopropylacrylamide, PEG, PEI). They can encapsulate hydrophilic/ hydrophobic drug moiety and control release rate. They are biodegradable and have ability to conjugate with any targeting agent. Current approaches used for preparation of nanogels can be divided into (a) chemical synthesis by polymerization (copolymerization), (b) chemical cross-linking of polymeric chains, and (c) physical self-assembly of polymers. They have several advantages over the conventional polymeric nanoparticles such as, high drug loading capacity, control of burst release, no use of solvent in the synthesis process and no energy input is required such as high-pressure homogenization and sonication. Further if PEG is used in the preparation of nanogel, it can evade capture by the mononuclear phagocyte system, resulting in a longer circulation time in the blood compartment. This prolonged plasma half life will increases the chances of passive diffusion in the tumor interstitium, across the leaky and hyperpermeable BBB. It also allows the conjugation with targeting ligand such as peptide (11-13).
During the past few years, the LRP1 and LRP2 receptors have been exploited to target drugs to the brain. They are structurally identical to cell surface LDL receptor gene family. Both receptors are multifunctional, multi ligand scavenger and signaling receptors. Many substrates have been exploited for these receptors such as receptor associated protein (RAP), lipoprotein lipase (LPL), Î±2-macroglobulin (Î±2M) , where as melanotransferrin (or P97), amyloid-Î² precursor protein (APP), apolipoproteinE (apoE), Angiopep 1 are LRP 1 specific and AÎ² bound to apoJ and apoE, aprotinin, and very-low-density lipoprotein (VLDL) are more specific for the LRP2 receptor (14-17). Researchers have found that LRP receptors are highly expressed on BBB as well as human glioblastoma cells (18).
C. Preliminary results
BÂ´eliveau's group first reported that melanotransferrin/P97 was actively transcytosed across the BBB and suggested that this was mediated by the LRP1 receptor. Melanotransferrin is a membrane-bound transferrin homologue that can also exist in a soluble form and is highly expressed on melanoma cells compared with normal melanocytes. They have shown that transport of P97 requires energy and is concentration- dependent, suggesting receptor-mediated endocytosis. Their results strongly support that the transferrin receptor is not responsible for P97 transendothelial transport rather LRP receptor is involved in the brain and transferrin does not compete for this receptor. Another advantage of using P97 is its very low concentration in the serum (100 000-fold lower than transferrin (19, 20), which suggests that it could deliver P97-conjugate(s) directly into the CNS. After conjugation to melanotransferrin, the BÂ´eliveau's group was able to successfully deliver doxorubicin to brain tumors in animal studies (21).
Studies done by Kabanov et al. reports that distribution of nanogel-ODN complexes in brain is higher than the radioactively labeled ODN after i.v. administration in mice. The amount of the radioactivity in the brain was greatly increased compared to the free ODN. However, accumulation of the radioactivity in the liver and spleen was significantly decreased (12).
D. EXPERIMENTAL DESIGN AND METHODS:
D Specific Aims
D.1) Specific aim 1:Â
To prepare and characterize unmodified and surface modified docetaxal loaded nanogels.
a). Preparation of unmodified nanogels:
A novel technique for synthesis of controlled molecular weight of polymer with a narrow molecular weight distribution has been utilized for the preparation of polymeric material. Atom transfer radical polymerization (ATRP) will be utilized for the preparation of NH2-PEG-PNIPAM block copolymers. The unmodified nanogels will be prepared by method described by Kabanov et al. Briefly nanoparticles of PEG-b-PNIPAM will be prepared by addition of N, N'ethylenebisacrylamide (5 or 10 mol% of NIPAM) as a cross-linker in the monomer solution at 500C in H2O and H2O/THF (1/1, vol/vol) mixture. After quenching the reaction with small amount of HCl (aq), the reaction mixture was dialyzed with distilled water by using the dialysis membrane for 7 days before measurement of dynamic light scattering and TEM (fig.1) (22).
b). Preparation of surface modified nanogels: Nanogels will be Surface functionalized by melanotransferrin (SantaCruz biotechnology, inc.),which is specific for LRPR. First, modification of the MTf (75 nmol) will be carried out in the phosphate-buffered saline (PBS) for 40 min at 25Â°C following the addition of 8-molar excess of the SMCC reagent in DMF. The protein will be separated from excess of SMCC by gel-filtration on NAP-20 column and used immediately in reaction with activated Nanogel. In separate vial 2 ml of a degassed 1% aqueous polymer solution will be mixed with 50Î¼L of 0.5 mM solution of 2-iminothiolane and allowed to stand under argon for 40 min at 25Â°C. The thiol-Nanogel will be used directly in reaction with modified mTf solution. Both solutions will be mixed together under argon, adjusted to pH 7, and the mixture will be incubated for 5 h at 25Â°C. mTf-Nanogel conjugates were dialyzed in membrane tubes with cutoff 4 KDa against phosphate buffer overnight at 4Â°C, and freeze-dried (12).
c). Surface morphology: Scanning electron microscopy will be used for studying surface morphology. Freeze dried nanoparticles will be used for analysis. Nanoparticles were attached to a double-sided tape, spray-coated with gold-palladium at 0.6 kV, and then examined under the electron microscope.
d). Particle size: A dynamic light scattering technique will be employed to obtain the particle size of nanoparticles in triplicates. The polydispersity values will be measured.
e). Release Studies: Release studies will be performed by suspending 2 mg drug equivalent modified and unmodified nanoparticles in a 500Âµl release buffer in a dialysis tubes. These tubes will be kept in a 10ml vial containing 5 ml release buffer. The entire 5 ml will be replaced, at regular interval, with fresh phosphate buffer to maintain sink condition. Sample collected will be analyzed for the total concentration.
D.2) Specific aim 2:Â
To study the uptake and cell proliferation of both surface modified and unmodified nanogel on rat brain microvessel endothelial cells (RBMEC).Â
a) Uptake studies
Confluent and mature RBMEC monolayer grown on 12-well plates will be washed 3 x 10 min with DPBS. Two milliliters of predetermined concentration of modified and unmodified nanogels in DPBS will be added to each of the12 well plates and incubated for 1 hr. At predetermined time points cells will be rinsed and lysed using 0.3 M sodium hydroxide and 0.1% Triton X-100. Protein estimation of the lysed cells will be performed using Bio-Rad protein estimation kit. The cell lysate will then be extracted by adding 1:5 (vol/vol) of organic extraction mixture (methanol and acetonitrile 5:4). The suspension will then be subjected to centrifugation and the supernatant will be analyzed by suitable HPLC technique. Drug concentrations will be calculated as Âµg/mg/ml of protein (23).
b) Cell proliferation studies
The number of viable cells will be evaluated by the method described by Ganta et al. MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay (Roche Diagnostics GmbH, Germany) as well as hemocytometer and trypan blue exclusion analysis (23).
D.3. Specific aim 3:
To evaluate in vivo time and dose dependent concentration of docetaxal in the brain, following intravenous administrations of the nanogels, using brain microdialysis technique in an unconscious Wistar rat model
Brain microdialysis:. The probe will be perfused with an artificial cerebrospinal fluid (140 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 1.2 mM Na2HPO4, 0.3 mM NaH2PO4, 3 mM glucose, pH 7.2) at a flow rate of 2ÂµL/min for one hour for equilibration. After equilibration, modified or unmodified nanogels will be given by intravenous/intracarotid route. One hundred microlitres of blood samples will be collected at predetermined time points from the jugular vein to determine plasma level. Dialysate will be collected every 20 minutes to study the brain concentrations of the drug. The samples will be analyzed with LC-MS. Animals will be kept under anesthesia for the entire duration of the experiment and euthanized under deep anesthesia by an over dose of sodium pentobarbital at the end of an experiment (26).
D.4. Specific aim 4:
To evaluate in vivo time and dose dependent tumor size reduction, in Wistar rats with brain-implanted C6-glioma, with nanogels
a) Animal model. Rat glioma cells (Brain Tumor Research Center, University of California, San Francisco, CA) were grown as monolayers in minimal essential medium. Cells will be harvested and resuspended for tumor implantation at a concentration of 105/5 ÂµL. Tumors will be induced in male Fischer 344 rats weighing between 125 and 150 g. Briefly cells (105) will be implanted in the right forebrain at a depth of 3 mm through a 1-mm burr hole. The surgical field will be cleaned with 70% ethanol, and the burr hole was filled with bone wax to prevent extracerebral extension of the tumor. Animals will be imaged using MRI beginning at 12 days after cell implantation for in vivo studies (24).
b) In vivo therapeutic brain tumor studies. Rats will be divided into four different groups, which consisted of controls (n = 5), Drug alone only (n = 9), Unmodified (n = 9), and modified nanogel (n = 9). Animals will be imaged every 2 to 3 days to follow changes in tumor diffusion changes for up to 2 weeks (24, 26).
c) Biodistribution studies: Biodistribution of the nanogel will be studied by administration of drug alone, unmodified and modified nanogels. Blood, ovary, heart, liver, kidney, spleen, lungs and tumor tissues will be collected after sacrificing the animal by of pentobarbital at 1, 3, 6, 12, & 24 h post dosing (25, 26).
Pitfalls and alternative approaches:
In our opinion we may have two pitfalls in this approach. First is the loading efficiency of the drug. And second is the effective conjugation of melanotransferrin to nanogels Former could be alternatively handled by changing monomer or preparation method of nanogel to suitably hold the drug. In case of LRPR if the melanotransferrine conjugation is not effective than we may surface adsorb the low density lipoprotein or conjugate peptide. Also coating with Polysorbate-80 has shown the targeting efficiency of nanogel towards the LRPR.