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A brain tumour is an intracranial solid neoplasam, a tumour within the brain or the central spinal canal. They are created by an abnormal and uncontrolled cell division, usually in the brain itself, but also in lymphatic tissue, in blood vessels, in the cranial nerves, in the brain envelopes (meninges), skull, pituitary gland, or pineal gland. Within the brain itself, the involved cells may be neurons or glial cells ( which include astrocytes, oligodendrocytes, and ependymal cells). Brain tumours may also spread from cancers primary located in other organs (metastatic tumours).
Glioblastoma Multiforme (GBM) is the most common and most aggressive malignant primary brain tumour in humans, involving glial cells and accounting for 52% of all functional tissue brain tumour cases and 20% of all intracranial tumours. Since GBM differs from the other cancers by its diffusive invasion of the surrounding normal tissue, it is impossible to make the complete removal of the tumour by the conventional surgical method and tumour recurrence from residual tumours is very possible. According to the pathological conditions of glioma in different grades, the different strategies should be taken to the design of the drug delivery system targeting glioma. When the glioma is still low grade , the BBB is intact and the drug delivery system should be able to cross the BBB and further target to glioma. However, when it develops to advanced -grade, drug delivery system can accumulate in the glioma by the enhanced permeability and retention (EPR) effect.
Polymersomes are novel drug carriers attained great attention in a recent days. The novelty in loading carboplatin in this delivery system increases in vivo stability, extends its blood circulation time , and further it will be releasing in a controlled manner at target site. The polymersomes having PEG as hydrophilic block possess "stealth" character which makes them invisible to immune system and enhance circulation time in the body.
Polymersomes targeting to brain tumer (novely compared to existing targeting aspects)
SYNTHESIS OF BLOCK CO-POLYMER
The dried Î±-methoxy-Ï‰-hydroxy-poly(ethylene glycol) (800mg, 0.27 mmol) was weighed in to a dry flask and Îµ-caprolactone (different amounts according to different M/I ratios) was subsequently added. The reaction mixture was stirred for 5min at 130Â°C in a preheated oil bath before the catalyst (stannous octoate, 1 drop) was added and the polymerization was performed for 3 h. The resulting viscous solution was rapidly cooled, upon which it is solidified. The crude polymer was dissolved in dichloromethane and precipitated in to heptane. The isolated yield were in the order of 90-95%.
CHARACTERIZATION OF BLOCK COPOLYMERS
1H-NMR spectra were obtained with CDCl3 as solvent and TMS as internal standard, using a Bruker AM 300 apparatus at 25Â°C.The actual PEG content of PECL was calculated from the integral height of hydrogen shown in 1H-NMR. IR spectra were recorded on a Nicolet MX-1 IR spectrometer. The samples were prepared by casting films from acetone solution on to KBr plates. Average MW and it distribution were determined by gel permeation chromatography (GPC, waters ALC/GPC 244, USA) operating with THF and calibrated with polystyrene standards. Intrinsic viscosity was measured with an Ubbelhode viscometer on 2.0% (g/dl) solution of polymer at 30Â°C in benzene, and the molecular weight was calculated using the equation : [È ]=9.94Ã-10â€’5Mv0.82. The transition temperatures of polymers were measured by DSC under a flow of nitrogen at a scanning rate of 10Â°C/min. The thermograms covered from -80 to 80Â°C.
PREPARATION OF NANOPOLYMERSOMES
The nanopolymersomes were prepared by the Nanoprecipitation method. Certain amount of block polymer and drug are dissolved in an organic solvent, which is a good solvent for all the blocks present , followed by hydration of the solution. The hydration can be done by either slowly adding water to the organic polymer solution or by injecting the organic solution in to water under magnetic stirring. The NPs are formed by making organic solvent to evapourate and washed twice and then centrifuged.
SURFACE MODIFICATION OF NANOPOLYMERSOMES WITH HUMAN TRANSFERRIN (Tf)
After centrifugation of the nanopolymersomes suspension, the pellet was further resuspended in a transferrin solution of 10 mM HEPES buffer with 150 mM NaCl (PH 7.4) for overnight. The surface -conjugated PEG-PCL nanoparticles were recollected by centrifugation under the same conditions. The excess Tf was removed by washing and centrifuging the nanoparticles for two times.
PARTICLE SIZE DISTRIBUTION AND ZETA POTENTIAL
The Nanpolymersomes size (diameter,nm), Polydispersity index and surface charge (zeta potential, mV ) were determined using a Zetasizer Nanoseries Nano-ZS (Malvern Instruments Ltd., Malvern, UK).
TEM (TRANSMISSION ELECTRON MICROSCOPY)
The morphology of NPs was confirmed using a transmission electron microscopy after negative staining with sodium phosphotungstate solution (2% w/w).
AFM (ATOMIC FORCE MICROSCOPY)
The shape and surface morphology of NPs were investigated by atomic force microscopy (AFM).
DIFFERENTIAL SCANNING CALORIMETRY (DSC)
The physical state of carboplatin loaded in the NP freeze dried and pristine carboplatin was investigated by DSC.
DRUG LOADING CONTENT (%)
The drug loading content was calculated by using the following equation :
DLC (%) = Amount of drug in NP/Amount of feeding polymer Ã- 100
ENCAPSULATION EFFICIENCY (%)
The encapsulation efficiency was obtained by using the following equation :
EE (%) = Amount of drug in NP/ Amount of loaded drug Ã- 100
INVITRO RELEASE STUDIES
The release profiles of carboplatin from PEG-b-PCL polymersomes were investigated at 37Â°C in different media, i.e, (a) acetate buffer, PH 5.0 (b) phosphate buffer, PH 7.4. The PH of above prepared carbolatin loaded polymers was adjusted to 5.0 using acetate buffer and to PH 7.4 using phosphate buffer and immediately transferred to dialysis bag with a MWCO of 12000-14000. The dialysis bag was immersed in to 50 ml of corresponding buffer at 37Â°C. At desired time intervals , the withdrawn buffer was replaced with fresh buffer. The samples were analyzed using UV-Visible spectroscopy at specific wavelength.
INVITRO CELLULAR UPTAKE OF NANOPOLYMERSOMES
Fluorescent nanopolymersomes were prepared by incorporating coumarin 6/FITC instead of drug in the formulations. Twelve-well plates were seeded with 105 U87MG cells per well and the cells were incubated at 37Â°C for 24h to allow cell attachment. After 24h, the medium was replaced by coumarin 6/FITC labelled NPs for 30 min, 1h and 2h. After incubation the NPs were removed and the wells were washed with ice-cold PBS and then visualized under fluorescent microscope.
INVITRO CYTOTOXICITY STUDY
Comparitive cytotoxicity of carboplatin in the formulation of Nanopolymersomes , transferring conjugated NPs, and marketed formulation was determined by MTT assay. MTT assay was carried out by using the U87MG (human glioblastoma) which express transferrin receptors. The cell line is cultured in a medium containing Dulbecco's Modified Eagles Medium(DMEM) and 10% foetal bovine serum. The U87MG cells were transferred to a 48 well tissue culture plates at 5Ã-104 cells per well and 3 wells per each sample (triplicate). The culture medium was then replaced with 200Âµl of medium containing carboplatin in the formulation of NPs , transferred conjugated NPs and marketed formulation and incubated for 48 hr at 37Â°C. Then, 50Âµl of MTT stock solution (5mg/ml) was added to each well and the plate was incubated for 3 hr at 37Â°C. Medium was then removed and 250Âµl DMSO was added and shaked for 30 min to dissolve the blue formazan crystal converted from MTT. Cell viability was assessed by absorbance at 590nm measured on a microplate reader.
INVIVO PHARMACOKINETIC STUDY
Twenty four male Sprague-Dawley (SD) rats weighing 200Â±20g were randomly assigned to four groups for pharmacokinetic investigation. Group 1 and 2 receives an i.v injection of carboplatin formulation of NPs and transferring conjugated NPs, then group 3 receives marketed formulation of carboplatin and group 4 was set to be control. At time points of 0 (pre-dose), 5,15, and 30 min, 1, 2, 4, 8, 12 and 24h post injection , blood samples (0.5ml) were collected from the orbital vein and centrifuged at 1000Ã-g for 10 min to obtain plasma. The plasma was stored at -70Â°C prior to analysis by HPLC.
INVIVO BIODISTRIBUTION STUDIES
Male Sprague-dawley (SD) rats of 130-180 gm were randomly distributed in to four groups with four rats in each group. Group 1 and 2 receives an i.v injection of the marketed carboplatin formulation and carboplatin NPs while Group 3 receives an transferrin conjugated NPs formulations ,respectively. Group 4 was set as control. The formulations was subsequently administered through the lateral tail vein and all animals were regularly monitored for their general health conditions, clinical signs, stress, movement and activity or mortality. After 4h, small amount of blood was drawn from the rats in each group through tail vein. Following that, perfusion was done using 0.1 M ringer's solutionfor 10 min. Organs such as liver, spleen, kidney, heart and brain were then extracted. The samples were stored at -80Â°C until analysis.
INVIVO EFFICACY STUDIES
STABILITY STUDIES (AS PER ICH GUIDELINES)
Stability of formulations during storage includes physical and chemical aspects and include the preservation of initial particle size and prevention of degradation reactions. To study the effect of the long-term storage on the physicochemical properties of the final optimized carboplatin loaded nanopolymersomes they are stored at different sets of temperature and humidity conditions based on ICH guidelines. Then, the samples are analyzed for the colour, PH, particle size, polydispersity, zeta potential, drug content and invitro drug release profile.
The formulation was sterilized by autoclaving. Autoclaving was done at 121Â°C, 15 lbs for 20 min. Test for sterility was carried out for formulation by direct inoculation method. Fluid thioglycolate and soyabean casein digest medium inoculated with Bacillus subtilis and Candida albicans were used in this study.