In-vitro Investigation of Polymeric Lipid Hybrid Nanoparticles (Plhns) Of a Chemotherapeutic Drug for the Treatment of Glioblastoma Multiforme

30 PHLNs (polymeric lipid hybrid nanoparticles) are core–shell nanoparticle structures made up of 31 polymer cores and lipid shells that have properties similar to both polymeric nanoparticles and 32 liposomes. Methotrexate (MTX) loaded PLHNPs containing tween 80, phosphatidylcholine, poly 33 D, L-lactic-co-glycolic acid (PLGA) & glyceryl tripalmitate prepared using solvent injection & 34 homogenization method for glioblastoma treatment option. The MTX loaded PLHNPs optimized 35 by Box–Behnken design to minimize particle size, higher entrapment efficacy, and maximize 36 MTX concentration in the brain at 4h. The particle size, entrapment efficacy, concentration of 37 drug in brain at 4h, zeta potential and AUC(Brain)/AUC(Plasma) ratio were in the range of 173.5138 233.37nm, 70.56-86.34%, 6.38-12.38 μg/mL, 25.78-36.31mV & 1.02-5.32. in-vitro drug release 39 studies, cellular internalization of optimized formulation against U-87 MG shows good anticancer 40 effects. 41 42

PLGA (Poly (D, L-lactide-coglycolide)) was used to improve the drug profile in this study [19]. 134 Furthermore, PLGA-encapsulated nanoparticles protect medications from degradation and aid 135 absorptive transcytosis into the endothelial cortex [20]. The reticuloendothelial system is 136 restrained from PLGA solid lipid nanoparticles below 200nm (RES) [21]. As a result, the primary 137 aim of this study was to make drug-loaded polymeric-lipid hybrid nanoparticles (PLHNPs) for 138 better stability and rigidity of the formulation [22]. 139 It's often challenging to get the correct medicament for glioblastoma or glioma (GBM) treatment. target specificity, and degrade more quickly, resulting in poor bioavailability. Polymeric solid 147 lipid drug distribution through the intravenous (IV) route would be the best way to solve both of 148 these issues, as polymeric lipid particles (>200nm) will penetrate the systematic circulation and 149 in PLGA matrix lipidic shell coating over MTX will shield it from macrophagic absorption and 150 reticuloendothelial opsonization. As a consequence, MTX could enter the brain and be processed 151 by endothelial cells [26]. 152 As compared to tablets, the key benefits of PLHNPs are increased bioavailability, sustained-153 release properties, and less side effects.Therefore, this investigation aimed to emphasize 154 formulation and in-vitro investigation of of MTX loaded PLHNPs using Box-Behnken design for 155 potential GBM treatment option. To obtain the prime objective of this study a single step synthesis 156 was performed where MTX loaded PLHNPs were synthesised using simple solvent injection 157 method. The polymeric-lipid hybrid nanoparticles (PLHNPs) comprising of PLGA in core and 158 glyceryl tripalmitate & phosphatidylcholine in shell. To stabilize and maintain rigidity, polyvinyl 159 alcohol (PVA) was added into PLHNPs formulation (Fig.1) to strengthen and maintain rigidity. 160 The research also sought to determine the physical compatibility (FTIR, DSC, XRD), particle 161 size, zeta potential, entrapment efficiency, morphology (TEM), in-vitro drug release, brain uptake 162 potential & pharmacokinetics studies, anticancer activity against U-87 MG glioma cells, cellular 163 uptake, biocompatibility (Lactate dehydrogenase assay, platelet aggregation, haemolysis) studies 164 of synthesised PLHNPs.

Optimization using Box-Behnken design:
198 Preliminary screening's key objective was to evaluate the various factors influencing particle size, 199 zeta potential, and trap efficacy of prepared nanoparticles. The most significant factors were the 200 concentration of phosphatidylcholine, glyceryl tripalmitate, and PLGA. The design of Box-201 Behnken[28]; where quadratic reaction surface design system RSM was introduced, the influence 202 of three factors (phosphatidylcholine, glyceryl tripalmitate, and PLGA) on the dependent 203 variables, i.e. particle size(Y1), zeta potential(Y2) and entrapment efficacy(Y3), the concentration 204 of drug in the brain at 4h interval(Y4), AUC(Brain)/AUC(PPC) (Y5) was evaluated. In total, 205 thirteen experimental runs were conducted and nanoparticles were prepared randomly to avoid 206 possible unreliable outcomes. Table 1 outlined the experimental design data. Model-fitting 207 analysis was performed using programme design-expert. 208 2 Evaluation and characterization of solid lipid nanoparticles: 209 2.1 Particle size analysis and zeta potential determination:

210
The average particle size and zeta potential were determined using Delsa Nano C (Beckman 211 Coulter, USA) [29]. Where nanoparticles were suspended in HPLC water and analysed through 212 triplicate photon correlation spectroscopy[30]. Measurement of particle size in Delsa Nano C at 213 a scattering angle of 90ᵒ at 25ᵒC for 120 cycles. For zeta-potential measurement, 0.5mL of the 214 sample was put in the electrophoretic flow cell and measurement was taken for 80 cycles. 215

216
The prepared nanosuspension was initially centrifuged at 4ᵒC for 30 min at 15,000 rpm. During 217 centrifugation, an identical formation of pellets can be seen within the centrifuge tubbings at the 218 rim. By means of decantation the supernatant was obtained and analysed using the Perkin Elmer 219 LAMBDA XLS Spectrometer in UV Visible spectroscopic method in triplicate at 244nm. The 220 blank was taken as the supernatant nanoparticular which was prepared without any drug [31]. To 221 obtain regression equation y=0.056x+0.18 (co-efficient of correlation R2=0.0997), the normal 222 calibration curve of Methotrexate between absorbance (y) and concentration (x) was plotted. The 223 following equation was used to calculate percentage drug entrapment efficacy: Trial tube (10,000-12,000Dalton 50mm) [32]. The dialysis tubes were preloaded with 229 nanoparticles that must have 2.5 mg of methotrexate and 2 mL of phosphate buffer solution before 230 being clamped with the USP Type II dissolution apparatus (pH 7.4). The paddle was rotated at 231 100 rpm, and the dissolution medium was known to be a 500 mL (pH7.4) phosphate buffer 232 solution while retaining 37 ± 2.5 °C. The aliquoted sample quantity (5mL) was removed from the 233 dissolution medium during perforation of the examination, and the same amount of freshly 234 formulated buffer solution was added to the dissolution medium. The material was analysed 235 spectrophotometrically at 244nm in triplicate. In addition, triplicate breakup experiments were 236 also carried out, and the results were expressed in terms of percentage of opioid release ± standard 237 deviation. For dissolution purposes the pH 7.4 phosphate buffer solution was used to maintain the 238 sink condition. and put on a carbon-coated grid (300 mesh). 278 2.9 Determination of brain uptake potential:

279
The concentration of drugs in the brain and 4th hour AUC brain/AUC plasma administration must 280 be measured for optimisation purposes [38]. These two were known as response variables in 281 formulation optimization. For brain absorption trials of pure drug, optimised formulation, 282 placebo, control (normal saline), male Wister rats were divided into four groups of 3 animals each 283 group. Before experimenting, the animals were kept for fasting. Water feed was kept accurate; 284 however, pure drug (MTX), test formulation, was administered during intravenous (IV) testing. 285 Blood samples(0.5mL) were obtained in heparin-containing tubes. After extracting blood samples 286 in various time intervals, i.e., 30, 60, 120, and 240 min, the samples were centrifuged for 20 min 287 at 13000 rpm, and plasma samples were collected and processed at -48ᵒC. Samples were for 288 analytical purposes. 289 By decapitation, animals were executed after extracting blood samples in the presence of 290 anaesthesia. Carefully removed brains and washed blood with bottling sheets. Brain tissues were 291 homogenised with a 0.01M phosphate buffer (pH 7.4) in tissue homogenizer (f NS-52 Tissue 292 Homogenizer, Microtec, Japan) for 15 min and centrifuged at 13000 rpm. The resultant 293 supernatant was deposited at -30ᵒC. 294 The amount of drug in brain samples was measured using HPLC. During the trial, 100μg/mL 295 methotrexate (20μL) was taken and dissolved in 2M sodium hydroxide solution and mixed 296 properly. A plasma/brain sample of 300μL poured the mixture. The mixture was thoroughly 297 vortexed, applying ethyl acetate, and 30 minutes shaking at 15ᵒC. The process was repeated three 298 times, the constituents' supernatant dried and reconstituted 400μL mobile phase. To quantify the 299 volume of drug present in plasma and brain, correctly tested HPLC methods are used. The samples 300 were properly screened using a 0.22μm syringe filter and the requisite sample quantity was 301 injected into the well-equipped Waters Alliance 2695 HPLC System. Methotrexate HPLC was 302 determined in 303nm with a flow rate of 1.5mL/min. Mobile phase consisted of buffer and 303 acetonitrile (98:2). Dissolving 27.22 g potassium dihydrogen orthophosphate in 1000 mL purified 304 water prepared the Buffer solution. During the HPLC method, the pH with NaOH solution was 305 changed to 6.0±0.05 and the resulting mixture was filtered using a 0.2μ nylon membrane filter.The 306 propensity for brain and plasma medication was measured by testing the drug in brain and plasma. 307 Also, AUC brain/AUC plasma was measured using the trapezoidal method.

334
On the basis of a previous publications, the drug, optimised formulation, and placebo were 335 analysed. Since the drug can be absorbed from the blood capillaries and, as a result, possibilities 336 of haemolyse blood cells after ingestion is high. Therfoer, a haemolysis analysis is needed to 337 support the product's biocompatibility. Hemocompatibility tests will also provide us an 338 understanding about how the drug, final product, and placebo will respond to blood [40]. 339 Blood was obtained from the nearest blood bank to conduct haemolysis tests [41]. The blood 340 samples (2.5 mL) were centrifuged for 15 minutes at 1345xg at room temperature. The graduated 341 centrifuge tubes used in this experiment were previously sterile. Micropipettes were used to 342 scrape the plasma coating during centrifugation. The developed erythrocyte pellets in the bottom 343 of the graduated centrifuge tubes were diluted and suspended further using normal saline solution. 344 At room temperature, the suspended part was centrifuged against 1345xg for 15 minutes. 345 was further diluted with 10mL saline water, and then 10, 50, and 100 g/mL of pure medication, 347 optimised dosage, and placebo were each combined with 2mL of erythrocyte suspension in sterile 348 tubes separately. The positive regulated examination, which fully lyzed erythrocytes, was 349 rendered by dissolving 1% triton X-100 in erythrocyte solutions. The negative control test, in 350 which erythrocytes are not lysed, was made by suspending erythrocytes in regular saline solution 351 in previously sterile Eppendorf tubes. The samples were incubated for at least 15 minutes at 37°C. 352 Nearly 200µL samples were removed at specific pre-determined time intervals, i.e., 0.5, 1, 2, 4,5, 353 6,7,8, 9, and 10 hr, thus centrifuging at 1345xg for 15 minutes. A 100µL supernatant was 354 incubated for 45 minutes at room temperature to achieve adequate haemoglobin to 355 oxyhaemoglobin conversion. At 580 nm, the absorbance was measured spectrophotometrically. 356 The following formula is used to calculate the proportion of haemolysis: 357 Where, A sample is the absorbance of the test sample containing the drug nanoparticles. A 359 spontaneous control is the absorbance of erythrocytes that are previously incubated with saline water. 360 On the other hand, A positive control was the absorbance of the supernatant of erythrocytes which 361 was exposed with 1% Triton X-100 solution made up of normal saline. The experiment was 362 performed in triplicate, and the data were expressed in mean ± SD (n=3). 363

Evaluation of erythrocyte membrane integrity:
364 Lactate dehydrogenase (LDH) is an enzyme that is produced by erythrocytes and can be 365 determined photometrically with the LDH assay kit / Lactate Dehydrogenase Assay Kit 366 (Colorimetric) (ab102526) [42,43]. The erythrocyte suspension was prepared according to the 367 protocol previously discussed on haemolysis. 1mL of erythrocyte suspension was used to 368 administer the drug's optimised formulation, the drug, and the placebo. The positive control 369 sample (100 percent lysed erythrocytes) was made by diluting the erythrocyte suspension with 370 1% Triton-X-100, while the negative control sample was made by diluting the erythrocyte 371 suspension with normal saline solution. To allow LDH natural preparation, 150/UL Lactate 372 dehydrogenase (LDH) was incubated with erythrocyte suspension at 37ᵒC. During specific time 373 periods, such as 2h, 4h, and 8h from suspension, 400L samples were extracted and independently 374 centrifuged at 1345xg for 20 minutes. LDH was detected at 500nm after the supernatant was 375 prepared with a ready-to-use LDH solution. The sum of LDH was calculated using the formula 376 below. . Before platelet counting, 2mL of blood was incubated with 387 different drug concentrations for 3 hours at 37oC, i.e. 10, 50, and 100 g/mL. After incubation, the 388 required volume of sample was diluted with standard saline water, and additional samples were 389 examined using a triplicate haematological counter (mean SD; n=3). It is important to do a 390 detailed study of platelet aggregation, which can be performed with an optical microscope. In this 391 experiment, heparinized blood samples were treated with various samples. After treatment with 392 the samples, peripheral blood smears were mounted on a glass slide and air-dried for 5 minutes. 393 With the aid of Leishman's dye, the blood smears were pigmented for 10 minutes. The stain was 394 then washed with purified water and covered with a glass cover. The staining essence of platelets 395 was examined using an optical microscope, and high-resolution images were captured. 396 3 Results and discussion: 397 3.1 Factorial design for optimization: 398 As seen in Table 1, a total of thirteen formulations (F1-F13) were created, and the optimization 399 method was developed using the Box-Behnken design. The influence of different independent or 400 input variables such as phosphatidylcholine concentration (X1), glyceryl tripalmitate (X2), and 401 PLGA quantity (X3) on dependent or outcome variables such as particle size (Y1), zeta potential 402 (Y2), and entrapment efficacy (Y3), brain drug concentration at 4h interval (Y4), and 403 AUC(Brain)/AUC(Plasma)(Y5) was investigated. Fig. 2(A&B) applies to the outcome of the 404 results . In the F1 to F13 batches, the mean particle size of the prepared nanoparticles ranged from In Fig. 3[I], the influence of all independent variables on particle size (Y1) was addressed (a). 419 The steep slope or curvature of factor 'C' or PLGA in Fig.3[I](a) suggests that concentrated PLGA 420 will have a greater impact on particle size. Increased phosphatidylcholine concentration reduces 421 particle size, as seen in the 3D surface plot of Fig. 3[I] concentration has a greater effect on zeta potential, according to the perturbation plot (Fig.3

[II](a) 431
This may be attributed to the fact that phosphatidylcholine is a surfactant. It was verified by 432 The perturbation plot (Fig.3[III](a) reveals that PLGA; factor "C" has a steep slope, suggesting 445 that PLGA concentration has a greater effect on entrapment efficacy. As can be seen in The perturbation plot (Fig. 3[IV](a) shows that PLGA; factor "C" has a steep slope, meaning that 455 PLGA concentration has a stronger effect on drug concentration in the brain at the 4h interval. 456 According to Fig.3[IV](b), glyceryl tripalmitate has no impact, whereas elevated 457 phosphatidylcholine concentration has an agonistic effect on drug concentration in the brain at a 458 4h interval; this may be attributed to the role of phosphatidylcholine enhancing the active 459 transport function of the drug in brain endothelial cells. However, it was clear from Once again, the perturbation plot (Fig.3[V](a) showed that factor "C" has a steep slope, 466 suggesting that PLGA Concentration will have a stronger impact on the Effects of AUC 467 (Brain)/AUC (Plasma) (Y5). According to Fig.3[V](b), glyceryl tripalmitate has no significant 468 effect on AUC (Brain)/AUC (Plasma); however, an increase in phosphatidylcholine concentration 469 could increase drug AUC in the brain; this could be due to the presence of the choline group, 470 which helps drugs cross the blood-brain barrier by speeding up acetylcholine synthesis and 471 boosting cognitive function of the brain. According to Fig. 3[V](c-d) and the polynomial 472 equation-5, PLGA improves medication transport in the brain and has a better brain-protective 473

impact. 474
As a result, increasing the PLGA concentration improved the AUC (Brain)/AUC (Plasma) ratio. 475 Furthermore, the positive and negative signs of the interaction terms, as well as the co-efficient 476 of the key outcomes, were used to denote the synergistic and antagonistic effects of the result 477 variables in the polynomial equation 1-5. The estimation of the p-value and F-value (Table 2) 478 often aids in determining the model's importance. The p-value was calculated at a 95% confidence level (0.05). The R 2 value and modified R 2 value of a model must be close to 1 in order for it to 480 be relevant. Furthermore, the lowest Bayesian Information Criterion (BIC) and Akaike 481 Information Criterion (AIC) must be used; the AIC aids in determining the optimal consistency 482 and goodness of fit standard. In comparison to another quadratic model of dependent variables, 483 To bring more specification in the optimization process by Design of Expert (Version 11.0) 488 software, the process capability index (Cpk) was identified in desirability studies (Fig. 4(a)). The 489 constraint variables were obtained, i.e., particle size at 193.143nm., zeta potential at 33.7515 mV., 490 entrapment efficiency 74.7937 %., Concentration of Drug in the brain at 4h interval at 491 8.80µg/mL., Effects of AUC(Brain)/AUC(Plasma) at 2.37169 respectively, when desirability was 492

493
From the overlay plot (Fig.4(b)) the desire region for constraining variables was identified within 494 the yellow design space. Considering overlay plot's independent variables 495 (phosphatidylcholine(X1) =130.025 & Glyceryl tripalmitate (X2) = 151.348) as a standard, three 496 batches were reproduced. The three batches' dependent variables or outcome variable's relative 497 standard error was found to be less than 9.00%. Therefore, the formula obtained from the overly 498 plot was considered as optimum. 499 Methotrexate release from the PLGA matrix by forming a canal. The zero-order kinetics was 508 detected as the polymer degraded. After 72 hours or 3 days, nearly 49.373.56% cumulative drug 509 release was reported, suggesting a continuous release trend of the nanoparticles, where the 510 correction coefficient (R 2 ) for the zero-order fit model was 0.9954 (Fig.5(a)). 511

512
To identify the chemical bonds and functional group heterogeneity within the samples, Fourier

Thermal characteristics:
531 Differential calorimetry scanning (DSC) was conducted to clarify the drug's crystalline and 532 amorphous behaviour. Often allows to identify drug association with polymer. Fig.5(c) showed 533 Methotrexate's DSC thermogramic peaks at 120.04ᵒC and 238.31ᵒC. The placebo endothermic 534 melting peak showed glyceryl tripalmitate melting point. PLGA's transition temperatures (Tg) 535 rose in placebo due to the presence of glyceryl tripalmitate, and two endothermic peaks were 536 found at 109.26ᵒC and 248.65ᵒC. In optimised PLHNPs, the PLGA melting point peak was 537 observed at 109.17ᵒC, but the peak intensity was poor compared to placebo, confirming optimised 538 PLHNPs amorphous nature. Additionally, the physical condition of the substance was probably 539 altered during encapsulation, so the formulation exhibits amorphous characteristics. 540

551
With the help of JEM-ARM300F GRAND ARM Atomic Resolution Electron Microscope, it was 552 possible to focus on drug-loaded PLHNPs, which were placed on cooper coated gride (300 mesh). 553 Fig.5(e) revealed that the projected particle was uniform in size and spherical in nature. The 554 projected nanoparticle in TEM analysis was found to have lesser particle size as compared to 555 particles size, which was obtained from the Delsa Nano C instrument (Beckman Coulter, USA); 556 this is because Delsa Nano C identifies the apparent volume of distribution of the diluted particles 557 in distilled water, however during TEM measurement, the particle may get dehydrated. Moreover, 558 the chances of aggregated particles while measuring in Delsa Nano C instruments was formidable, 559 as prior measurement, the nanosuspension was exposed to 15 minutes sonication. 560

561
A particular HPLC method was developed from the brain homogenates and plasma samples of 562 experimental animals, i.e. Wister rates, which appeared to have a high degree of specificity and 563 precision at a specific time interval. After 4 hours of drug administration, the AUC brain/AUC Plasma 564 drug concentration in plasma was measured. The drug concentration in the brain was estimated 565 to be 2.43µg/g after 4 hours of administration, and the AUC brain/AUC Plasma of the drug was found 566 to be 2.23 after 4 hours of administration. This implies the drug concentration in the brain was 567 2.23 times greater than the drug concentration in plasma, which was absorbed by intravenous 568 route. As a result, Methotrexate may be targeted in the brain in a passive manner. 569

Cytotoxicity studies against U-87 MG glioma cells: 570
To compare the efficacy of Methotrexate against U-87 MG glioma cell lines, cytotoxicity tests 571 were carried out. Methotrexate, placebo-PLHNPs, and optimized-PLHNPs cytotoxicity against 572 87 MG glioma cell lines can be seen in Fig.6 (a). In all concentrations, the cellular cytotoxicity 573 of Methotrexate loaded Optimized-PLHNPs was found to be significantly higher than that of free 574 Methotrexate (p0.05). The cytotoxicity study's most unexpected result was that placebo-PLHNPs

579
The U-87 MG glioma cells were picked up with optimized-PLHNPs, and stained with DAPI 580 reagent, as shown in Fig.6(b). Suitable filters were used to capture the CLSM representation of 581 DAPI reagent. After 4 hours of incubation with U-87 MG glioma cells, the image indicates 582 extensive internalisation of optimized-PLHNPs, It's possible that nanoparticles can clump 583 together in the cytoplasm. Many apoptotic processes, such as the expression of caspase-3 mRNA, 584 the downregulation of metalloproteinases (MMPs), the upregulation of p53, and the deactivation 585 of IAP5, are believed to take place in the cytoplasm, according to various reports. As a 586 consequence, optimized-PLHNPs, may be an useful tool to induce cytotoxicity in the cytoplasm. 587

Evaluation of haemolysis:
588 The most important study to learn about the behaviour of prepared PLHNPs with blood was an 589 in-vivo toxicity study. The association of PLHNPs with blood and its substances must be 590 investigated. The PLHNPs of PLGA, which are made up of lactide (LA) and glycolide (GA) 591 monomers, have a minor impact on homolyses. The findings of the analysis suggest that PLHNPs 592 can be used with care. The profile of percentage hemolysis absorbance with 0.5th and 10th-hour 593 shifts of erythrocytes with different treatments (10,50,100g/ml) containing Methotrexate, 594 placebo-PLHNPs, and Methotrexate primed optimised PLHNPs is depicted in Fig.7(a-d). It can 595 be inferred from Fig.7(d) that placebo-PLHNPs and Methotrexate-loaded optimised PLHNPs 596 cause fewer haemolysis than Methotrexate. Regardless of Drug and PLHNPs concentrations of 597 10,50,100g/mL, the placebo-PLHNPs and Methotrexate-loaded optimised PLHNPs displayed 598 fewer hemocompatibility (>1%) and were found to be safe to RBC membrane integrity. Most 599 notably, Methotrexate-loaded optimised PLHNPs formulations display fewer haemolysis; this is 600 attributed to adequate Methotrexate encapsulation inside the PLHNPs, which prevents red blood 601 cells from Methotrexate-induced haemolysis. One thing that can be proven from the haemolysis 602 studies is that i.v. administration of Methotrexate primed optimised PLHNPs can have a beneficial 603 impact on erythrocytes. As a consequence, there's a decent risk of lowering malondialdehyde 604 (MDA) levels and carbonyl group content. As a consequence, methotrexate-loaded optimised 605 PLHNPs can prevent erythrocyte degradation during glioma care when delivered intravenously. 606

Evaluation of erythrocyte membrane integrity:
607 White blood cells and blood plasma were separated using centrifugation, and natural saline 608 comprising erythrocytes was used instead. Enumerating the LDH enzyme is expected to 609 determine membrane integrity. Only when the structural integrity of the erythrocytes was 610 disrupted was a hyper or elevated degree of LDH recorded. Figure 8(a-c) shows the sum of LDH 611 released after treatment with 2mL of erythrocyte suspension with Methotrexate, methotrexate-612 loaded optimised PLHNPs (equivalent to 10, 50, and 100 g/mL of Methotrexate), and placebo-613 PLHNPs. By matching placebo-loaded optimised PLHNPs of 10, 50, and 100 g/mL concentration 614 at 8th-hour intervals to Methotrexate-loaded methotrexate-loaded optimised PLHNPs of 10, 50, 615 and 100 g/mL concentration, the LDH release was not as conspicuous (20%). Triton X 100, on 616 the other side, displays approximately 290 % LDH release, suggesting total erythrocyte 617 annihilation. As a result, methotrexate-loaded optimised PLHNPs are unlikely to damage 618 erythrocyte membrane integrity.Thus, methotrexate-loaded optimised PLHNPs will be suitable 619 for intravenous administration. solution, which postulating Methotrexate did not show any platelet aggregation ( Fig.9(a) PLHNPs, and Methotrexate loaded optimized PLHNPs was shown in Fig.9(b). 639 4 Discussion:

642
The present study demonstrated the in-vitro investigation and systematic optimization of 643 Methotrexate loaded PLHNPs using Box-Behnken design. The optimization process seeks to 644 provide adequate particle size, zeta potential, entrapment efficacy with maximum drug ratio in 645 the brain. From the optimization process, it was also understood that the increased concentration 646 of PLGA and glycerol tripalmitate helps to increase the particle size.On the other hand, increase   Methotrexate-loaded PLHNPs is synthesised using one-step solvent injection method. With 696 higher drug content in the brain, PLHNPs with a lipid shell and a polymeric core display less 697 hemolysis and platelet aggregation. It can be concluded that phosphatidylcholine and glyceryl 698 tripalmitate coated with PLGA core matrix can open up a new way to deliver lipophilic 699 constituents with increased potential for glioma treatment. 700 6 Acknowledgment: