Cationic bovine serum albumin (CBA) conjugated poly lactic-co-glycolic acid (PLGA) nanoparticles for extended delivery of methotrexate into brain tumors

Abstract

Current strategies for the treatment of brain tumors have been hindered primarily by the presence of the highly lipophilic, insurmountable blood–brain barrier (BBB). The purpose of the current research is to investigate the efficiency of the engineered biocompatible polymeric nanoparticles (NPs) as drug delivery vehicles to bypass the BBB and enhance the biopharmaceutical attributes of anti-metabolite methotrexate (MTX) encapsulated NPs. The NPs were prepared by the solvent diffusion method, using cationic bovine serum albumin (CBA), and were characterized for physicochemical parameters, such as particle size, polydispersity index, and zeta-potential, while the surface modification was confirmed by FTIR, and NMR spectroscopy. The developed NPs exhibited the zestful relocation of FITC tagged NPs across the BBB in albino rats. Further, hemolytic studies confirmed them to be non-toxic and biocompatible, as compared to free MTX. The in vitro cytotoxicity assay of our engineered NPs on HNGC1 tumor cells proved the superior uptake in tumor cells, and elicited a potent cytotoxic effect, compared to plain NPs and the free MTX solution. The outcomes of the study evidently indicate the prospect of CBA conjugated poly (D,L-lactide-co-glycolide) (PLGA) NPs loaded with MTX in a brain cancer bomber, with the amplified capability to circumvent the BBB.

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1. Introduction

The treatment of brain tumors presents a complicated and unsolved problem because of the difficulty in bypassing the blood–brain barrier (BBB),^1,2 and the mitigation of brain tumors is still the foremost challenging obligation.³ The BBB, a highly lipophilic barrier, consists of the vascular structures composed of physical obstacles (tight junctions of astrocytes and endothelial cells), and well organised traffic controllers of the metabolic barriers (multidrug resistance (MDR) pathways), that manage the transport of bioactive materials across the BBB.^4–7 The cassette transport pump (p-glycoprotein) plays a fundamental role in the drug efflux at the level of the BBB.⁸ However, hydrophobic small molecular drugs can be easily transported across the BBB, and diverse attempts have been made to transport active molecules across.^9,10 The successful management of brain tumors necessitates bypassing, inactivating or suppressing the metabolic BBB barrier.¹¹ Therefore, the expansion of an efficient system to bear the active molecules across the BBB has been a major challenge in research, and efforts must therefore be resolute in developing an approach that enhances bioavailability in the central nervous system (CNS).

Nanotechnology has indeed exploited most of the pharmacological complications of active molecules that result in increased therapeutic efficiency.^12–15 Biocompatible nanostructured systems facilitate the interactions with biomolecules and the interior environment of the cell and may sidestep the highly lipophilic physical obstacle that limits the passage of a wide variety of therapeutic agents across the BBB.^16–18 Nowadays, surface engineering of nanocarriers for target specific delivery of active molecules has been considered a viable strategy for improving therapeutic efficacy in the management of brain tumors.^19–22 Thus, targeting strategies for crossing the barriers present in the BBB are being developed to overcome the limited transport across it. Of the various approaches, adsorptive transcytosis via cationic bovine serum albumin (CBA) has been scrutinized to bypass the BBB without influencing the integrity of the tight junction at the BBB.^23–25 Surface modification of NPs alters the BBB via adsorptive transcytosis, where interaction between BSA and the cell membrane at the BBB demonstrate that BSA preferentially encourages the transport of nanocarriers across the BBB.^26–29

Methotrexate (MTX), a dihydrofolate reductase (DHFR) inhibitor acting in the S-phase of the cell cycle plays a crucial role in the treatment of brain cancer by introducing itself within the folate stacks.^30,31 However, the inability to deliver MTX across the BBB, owing to its hydrophilic nature and the distribution of the drug at noncancerous sites has severely compromised its clinical application. Therefore, nanostructured drug delivery vehicles, which are able to bypass the BBB and cell membranes of brain tumor cells are an urgent and inevitable requirement for the treatment of brain tumors. The United States Food and Drug Administration (US-FDA) approved the biodegradable non-immunogenic, biocompatible polyester polymers such as poly (D,L-lactide-co-glycolide) (PLGA) in the targeted delivery of drugs for various ailments.^32–35

The prepared NPs formulation engineered with CBA was physico-chemically characterized and evaluated using in vitro, ex vivo (cell line) and in vivo studies. Moreover, a comparative study of brain uptake of FITC tagged NPs following intravenous injection was confirmed by fluorescence microscopy. In a nutshell, the present study with CBA modified PLGA-NPs encapsulated MTX opens new horizons, not only to understand the difficulties, but also to improve the management and effective treatment of brain cancer.

2. Materials, methods and instrumentation

2.1. Materials

MTX, bovine serum albumin (BSA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and (EDC) were purchased from Sigma Aldrich (Germany). PLGA (50 : 50), N-hydroxy succinimide (NHS), di-methyl-sulphoxide (DMSO), PluronicF-68 (PF-68), FITC and Triton-X100 were purchased from Himedia, Mumbai, India. Nylon membrane filter (0.22 μm) was purchased from Pall Gelman Sciences (USA). All other reagents and solvents were of analytical grade, unless otherwise specified. Ultra-pure deionized water was used throughout the study.

2.2. Preparation of cationic bovine serum albumin (CBA)

CBA was prepared from BSA following the well-established procedure reported earlier, with trivial modifications (Feng et al., 2009). Briefly, 0.5 g of BSA was treated with a small excess of ethylenediamine (EDA), previously solubilised in conjugation buffer (MES; 0.2 mol L⁻¹) at 4 ± 1 °C, while being agitated using a magnetic stirrer (Remi, India); the pH was adjusted to 4.7. Subsequently, 50 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were added and allowed to remain undisturbed for 2 h in the dark, at ambient temperature. The reaction was continued with the addition of 0.4 ml of acetate buffer (2 mol L⁻¹; pH 4.75). Finally, the solution was dialyzed extensively against ultrapure deionised water for 48 h, lyophilized and stored in a hermetically sealed container. BSA and CBA were further characterized using NMR (Bruker DRX, USA) and FTIR spectroscopy (Perkin-Elmer, USA).

2.3. Fabrication of nanoparticles

The MTX loaded PLGA NPs were prepared in accordance with the solvent-diffusion technique described by Song et al. in 2006,³⁶ with slight modifications. Briefly, MTX was dissolved in PBS (pH 7.4)/DMSO. Subsequently, an aqueous solution of Pluronic® F-68 (PF-68) (PBS; pH 7.4) was added as a stabilizer to the drug solution, and 1% w/v PLGA solution (the organic phase; 10 ml) in ethyl acetate was added drop-wise into 20 ml of an aqueous phase consisting of the drug and stabilizer, under regular magnetic agitation (REMI Instrument India). This pre-emulsion was maintained above the melting temperature using a hot water bath, and the high speed stirring was set to attain a pre-emulsion phase, followed by sonication. In addition, a measured volume of ultra-pure deionized water was supplied to the o/w emulsion under gentle stirring to facilitate the diffusion of the organic phase in an aqueous solvent, leading to the formation of MTX loaded PLGA NPs (MTX–NPs). Subsequently, the NPs dispersion was filtered through 0.22 μm membrane filters. Finally, the MTX–NPs dispersion was lyophilized (Heto Drywinner, Denmark, Germany) and stored for further studies. FITC labeled NPs were prepared by co-encapsulation of FITC along with MTX in PLGA NPs.

2.4. Fabrication of CBA conjugated MTX loaded PLGA NPs (CBA–MTX–NPs)

Freeze dried MTX–NPs were dispersed in ultra-pure deionized water at a concentration of 20 mg ml⁻¹. Subsequently, 50 mg of N-hydroxy succinimide (NHS) were added and allowed to be agitated for the next 6 h, followed by the addition of EDC (15 mg), while constantly stirring (Magnetic stirrer; Remi, India) for 48 h at ambient temperature. Afterwards, CBA (50 mM) was added, with further agitation (all night at 2000 rpm) to facilitate its conjugation with the MTX–NPs. The dispersion was then finally dialyzed against ultra-pure deionized water for 30 min to take away free CBA and other impurities, followed by lyophilization (Heto Drywinner, Denmark, Germany).

2.5. Characterization of nanoparticles

2.5.1. Measurement of particle size and zeta potential. The average particle size and poly dispersity index (PDI) of MTX–NPs and CBA–MTX–NPs was determined by a Zetasizer (DTS ver. 4.10, Malvern Instruments, England). Briefly, a nano-particulate sample dispersion was added to polystyrene cuvettes, diluted with ultra-pure deionized water and analyzed at a 90° fixed angle. The zeta potential of the NPs formulation was measured by determining the electrophoretic mobility with a laser-based multiple angle particle electrophoresis analyzer, Malvern Zetasizer (DTS Ver. 4.10, Malvern Instruments, England). The nanoparticles were suspended in ultra-pure de-ionized water and kept in an electrophoretic cell with an electric field of 15.24 V cm⁻¹ and the zeta potential was measured.

2.5.2. Scanning electron microscopy. The surface morphology was determined by scanning electron microscopy (SEM). In brief, the NPs powder was sprinkled on a double adhesive tape, which was stuck on an aluminium stub. Following this, the stubs were coated with gold at a thickness of about 300 Å by using a sputter coater. All NPs were examined under a SEM (LEO 435 VP, Eindhoven Netherlands) at an acceleration voltage of 30 kV and photographs were taken at various magnifications.

2.5.3. Encapsulation efficiency. The drug encapsulation efficiency was estimated using the dialysis method for separating unloaded MTX from the NPs. This was then analyzed spectrophotometrically to indirectly determine the amount of drug bound with the NPs. 5 ml of the MTX–NPs dispersion was placed into a dialysis bag (MWCO 1000 kDa, Himedia, India) and dialyzed against 50 ml of 0.1 N NaOH for 60 minutes with magnetic stirring (50 rpm). Samples (0.5 ml) were withdrawn occasionally and collected in HPLC vials (Himedia, India) until no drug was detected in the receptor compartment.³⁷ All samples (injection volume – 10 μl) were quantified for MTX content by HPLC at a flow rate of 1 ml min⁻¹ at 302 nm and at 40 °C. Samples were loaded into the HPLC system to quantify the unentrapped amount of MTX, so as to indirectly quantify the amount of entrapped MTX within the NPs.^38–40 The same procedure was carried out to measure the entrapped drug in CBA conjugated NPs and the quantification was performed using HPLC (Shimadzu, Japan). The reversed phase C₁₈ column (4.6 mm × 250 mm, 5 μm) was used for chromatographic separation. The mobile phase was a mixture of buffer (pH 6.0) and acetonitrile at a ratio of 9 : 1. The buffer consisted of 0.12 M di-sodium hydrogen orthophosphate and 0.03 M citric acid.⁴¹

2.6. In vitro release study

The ability of NPs to release drugs into external media was determined by conducting the in vitro drug release assay on the developed NPs. The dialysis tube diffusion technique was used to estimate the in vitro release of MTX from MTX–NPs and CBA–MTX–NPs. The fixed volumes of the prepared nano-particulate formulations (5 ml) were poured into the dialysis tubing (MWCO 1000 kDa), tied at both ends and immersed into receptor media consisting of 100 ml of phosphate buffer saline, pre-equilibrated at 37 ± 2 °C, followed by moderate stirring (magnetic stirring, 100 rpm). 0.5 ml samples from the recipient compartment were withdrawn intermittently and an equal quantity of fresh medium was added after each sample withdrawal in a receptor compartment to maintain a constant sink volume over the experiment. The same practice was adopted for the determination of the drug release profile at pH 5.4 (mimicking acidic tumor environment). Samples were analyzed by HPLC using the same method described above and the amount of MTX released over time was quantified.^41,42

2.7. Ex vivo study

2.7.1. Cytotoxicity study. Human brain cancer cell lines (C6 glioma) were purchased from the National Center for Cell Sciences (NCCS), Pune, India, and cultured in DMEM (Dulbecco's Modified Eagle's Medium, Himedia, India), supplemented with 10% v/v heat-inactivated fetal bovine serum (FBS), 1% v/v streptomycin and 3 mM glutamine in a 37 ± 2 °C humidified incubator and 5% CO₂ atmosphere. Cytotoxicity of the NPs was evaluated by sulforhodamine B (SRB) staining assay.⁴³ This assay is based on the measurement of optical density for the determination of cell survival and proliferation. Principally, this assay determines the metabolic activity of viable cells. Exponentially growing C6 glioma cells were plated at a density of 7 × 10⁴ cells per ml in 24 well plates (Sigma, Germany). The cells were then incubated with predetermined concentrations (100.0–0.01 μM) of MTX (free MTX; MTX–NPs, CBA–MTX–NPs and CBA [1 mg ml⁻¹] + CBA–MTX–NPs) under a restricted environment for 72 h. Afterwards, 20 μl of cold trichloroacetic acid (TCA; 50%) was added to each well and kept undisturbed for an hour at 4 °C to facilitate cell fixation. Subsequently, the plates were rinsed with ultra-pure deionized water, dried and stained with 50–60 μl of 5% w/v SRB reagent (constructed in 1% v/v acetic acid) for an hour. Then, 150 μl of acetic acid was aspirated to remove the excess of SRB reagent. In each well, Tris was added and well-mixed on a shaker for few minutes. The optical density (OD) of each well was then measured at 490 nm via microplate spectrophotometer (Model 680, Bio-Rad, Japan).

2.7.2. Cellular uptake assay. The cellular uptake efficiency of NPs was evaluated as a function of the ligand, using a fluorescence activated cell sorter (FACS) instrument (BD Biosciences FACS Aria, Germany) against C6 glioma cells. Glioma cells were cultured in accordance with the process described under cytotoxicity studies. Briefly, cells were seeded in 6 well plates (Sigma, Germany) at 2 × 10⁶ cells per ml, using fresh culture medium, and were suspended for 12 h in a humidified incubator at 37 ± 2 °C with a 5% CO₂ atmosphere. Cellular internalization of different doses of MTX formulation was determined by in vitro incubation of cells with fluorescein isothiocyanate (FITC), FITC labeled NPs and CBA [1 mg ml⁻¹] + CBA conjugated FITC labeled NPs in separate wells for 1 h. NPs that were adhered to the cell surface were detached by washing each well twice with PBS (pH ∼ 7.4). Further, cells were trypsinized (0.1% w/v), kept for 5 min, and harvested by adding 1 ml PBS (pH ∼ 7.4), followed by probe sonication five times, to obtain the cell lysate. Finally, the cell lysate was centrifuged at 12 000 rpm for 10 min, and the supernatant was subjected to fluorescence assay using FACS. The same procedure was followed to study the cellular uptake profile after 2 h and 6 h.^3,44

2.8. In vivo pharmacokinetic and biodistribution studies

The in vivo validation of the developed formulation (MTX–NPs and CBA–MTX–NPs) was performed to evaluate the CBA mediated transport while bypassing the BBB, and drug distribution in various organs of Balb/c mice (20–25 g; either sex). All animal experimentation procedures were carried out with prior permission and approval from the Institutional Animals Ethical Committee, Dr Hari Singh, Gour University, Sagar (M.P.; India). The animals were fasted overnight with free access to water ad libitum. A short incision was made through the skin to expose the cranium and approximately 6 × 10⁵ C6 glioma cells/10 μl in serum-free 2% v/v DMEM were stereotaxically rooted into the right forebrain of each animal.⁴⁵

Previously weighed tumor bearing animals were randomly divided into four groups of nine animals each. MTX, MTX–NPs and CBA–MTX–NPs were dispersed in normal saline. The formulations were administered at a dose of 5.0 mg kg⁻¹ body weight through the lateral tail vein of the animals of the first, second and third groups, respectively.⁴⁶ Animals of the fourth group served as a control for this study. Approximately 300 μl of blood samples were withdrawn from the retro-orbital plexus under mild anesthesia at 0.16, 0.5, 1.0, 2.0, 3.0, 5, 8, 12, 18, 21 and 24 h intervals in heparinized tubes. Blood samples were collected from three animals of each group, which were sacrificed by cervical dislocation after the study.

The organs viz. the brain, liver, kidney, heart, spleen and the tumor were isolated carefully, weighed and stored at −80 °C until experimentation. Afterwards, blood samples were collected from three other animals (n = 6) of each group for 2–8 h intervals; the animals were euthanized and different organs were isolated. This exercise was also repeated for the last three animals of each group for 8–24 h intervals.

Plasma was separated from blood samples by centrifugation at 10 000 rpm for 15 min. Afterwards, 100 μl of plasma was filtered into a micro-centrifuge tube, and the same quantity of acetonitrile was added to precipitate the protein.

The organs were chopped and homogenized to detach the tissues, vortexed for one minute, and kept undisturbed for 30 min. Tissue homogenates were then treated with 100 μl of acetonitrile. Further, the serum and tissue homogenates earlier admixed with acetonitrile were vortexed for 1 min, and subsequently centrifuged at 5000 rpm for 15 min. The supernatant was filtered through a 0.22 μm syringe filter, collected in HPLC vials (Himedia, India), and quantified for MTX content. Quantification of MTX was done in serum, as well as in various tissues viz. brain (glioma cells), heart, liver, spleen and kidney, performed by the HPLC method as described earlier.^41,47

2.9. Hemolytic toxicity

Hemolytic studies were performed following the procedure described earlier, with slight modifications.^39,48 Briefly, whole human blood samples were collected from a healthy person (with kind consent) and heparinized in HiAnticlot blood collection vials (Himedia, India). Subsequently, the red blood cells (RBCs) suspension was centrifuged and resuspended in normal saline. Two mL of the RBCs suspension were separately dispersed in normal saline solution, producing no hemolysis (which served as a control, ‘0% hemolysis’), and in distilled water, considered to produce 100% hemolysis. One ml of adequately diluted plain MTX solution, MTX–NPs, and CBA–MTX–NPs was incubated individually with 2 ml RBCs suspension and the volume was made up to 10 ml with normal saline.

The formulations were taken in such amounts that the resultant final concentration of MTX was equivalent in every case, so as to facilitate the assessment of the extent of hemolysis, followed by the effect of Tween 80 (Himedia, Mumbai, India) coating on hemolysis. Further, the formulations were allowed to incubate at 37 ± 1 °C for 30 min, followed by centrifugation at 4000 rpm for 10 min. Subsequently, supernatants were taken and diluted with an equal volume of normal saline and the absorbance was measured by UV-Visible spectrophotometer (Shimadzu, 1601 Japan) at 540 nm, against normal saline, diluted similarly as the blank. The percent hemolysis was then determined for each sample by taking the absorbance of distilled water, which was assumed to be producing 100% hemolysis.

2.10. Data analysis

Statistical analysis of the data was articulated as the mean ± S.D and statistical analysis, computed using one-way analysis of variance (ANOVA) with a Tukey–Kramer multiple comparison post-test using GraphPad InStat™ software (GraphPad Software Inc., San Diego, California). A probability level of p < 0.05 was considered to be significant.

3. Results and discussion

3.1. Preparation of CBA

The FTIR spectroscopy of pure BSA (Fig. 1A) shows the distinctive peaks at 3280.16 cm⁻¹ (stretching vibration of –OH), 2966.17 cm⁻¹ for amide (–N–H stretching vibration), 1635.01 cm⁻¹ (C=O stretching vibrations), 1515.49 cm⁻¹ (coupling of bending vibration of N–H and stretching vibration of C–N) cm⁻¹, 1388.88 cm⁻¹ (aromatic –C=C–) and 1037.46 cm⁻¹ (C–O bending) (Fig. 1A). After cationization, discrepancies in the FTIR peaks are understandable with elevated intensity at parallel wavelength because of the presence of auxiliary ethylene amine groups in CBA, incorporated during the cationization. Shifting of the FTIR peak of –OH groups, C=O stretching, and the disappearance of 1388.88 cm⁻¹ (aromatic –C=C-stretching) confirmed the cationization of BSA (Fig. 1B). In NMR spectroscopy, the amine group (–NH₂) shows a high shielding effect and the obtained spectra of CBA illustrated its characteristic peak at 3.5 ppm, which confirmed the presence of the amine group in CBA (Fig. 2A).

Fig. 1 FT-IR spectra of (A) BSA, (B) CBA and (C) CBA–MTX–NPs.

Fig. 2 NMR of (A) CBA and (B) CBA–MTX–NPs.

3.2. Preparation of CBA conjugated MTX loaded NPs (CBA–MTX–NPs)

The NPs were fabricated by an emulsification–diffusion method and the carboxylic groups of the NPs were conjugated to amine functionalities of CBA, according to a previously reported method, with slight modifications.^49–51 This technique involved the quick diffusion of solvent across the solvent–lipid phase into the aqueous phase, followed by the evaporation of solvent, leading to enhanced rigidity of the NPs.

Addition of CBA in the presence of NHS and EDAC, leads to the formation of an amide (–CONH–) bond between amine (–NH₂) groups of CBA and carboxylic acid (–COOH) present on the surface of the NPs. Amalgamation of CBA appended NPs was further confirmed by FTIR and NMR spectroscopy. The presence of peaks depicting N–H stretching at 3432.01 cm⁻¹, C=O stretching at 1723.95 cm⁻¹ and C–O stretching at 1360.42 cm⁻¹, confirmed the amide bond formation, and hence the conjugation between NPs and CBA (Fig. 1C). In the NMR spectra, the acidic group (–COOH) demonstrates its peak at deshielded positions, or away from the reference line of tetra methyl silane. In the NMR spectra of CBA–MTX–NPs, a distinguish peak with less intensity at 8.5 ppm indicated that most of the free carboxylic groups got saturated with the amine group of CBA, thus confirming the formation of amide bonds between carboxylic groups of MTX–NPs made up of PLGA and amine groups of CBA (Fig. 2B).

3.3. Characterization of nanoparticles

3.3.1. Size, zeta potential and surface morphology. The average particle size of CBA conjugated MTX–NPs as measured by the dynamic light scattering technique was found to be larger than that of the unconjugated formulation (Fig. 3 and Table 1). The disparity in the average size may be due to the conjugation of CBA on the surface of MTX–NPs. The average particle size of MTX–NPs was found to be 108.3 ± 3.1 nm, while in the case of the CBA conjugated formulation, it was 120.9 ± 4.4 nm. The mean diameters of NPs were well below 200 nm and these fallouts are in harmony with SEM photographs of the CBA conjugated NPs (Fig. 4A and B).

Fig. 3 Particle size of (A) MTX–NPs and (B) CBA–MTX–NPs. Zeta potential of (C) MTX–NPs and (D) CBA–MTX–NPs.

Table 1 Zeta potential, particle size, PDI and percentage drug entrapment of nano-formulations^a

Formulation code	Zeta potential (mV)	Particle size (nm)	PDI	% drug entrapment
a Results are presented as mean ± SD (n = 3).
MTX–NPs	−13.7 ± 0.4	108.3 ± 3.1	0.015 ± 0.004	79.9 ± 2.4%
CBA–MTX–NPs	−5.55 ± 0.3	120.9 ± 4.4	0.155 ± 0.007	71.3 ± 1.8%

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Fig. 4 (A and B) SEM images of CBA–MTX–NPs and (C) the in vitro release profile of MTX from MTX–NPs and CBA–MTX–NPs in PBS (pH 7.4) and PBS (pH 5.4). Inset graph represents the drug release at the initial time points. Each data point represents mean ± SD (n = 6).

Zeta potential is an imperative criterion in order to establish the storage stability of nanometric particulate systems. The zeta potentials of MTX–NPs and CBA–MTX–NPs formulations were found to be −13.7 ± 0.4 mV and −5.55 ± 0.3 mV, respectively (Fig. 3 and Table 1). The higher negative magnitude of the zeta potential might be ascribed to the –COO (carboxylic) group existing at the surface of polymeric NPs. The lowering of the zeta potential value upon CBA conjugation was reasoned to be due to the subsequent replacement of the –COO group with terminal amine (NH₂) groups after protein conjugation.⁵¹ The elevated magnitude of the zeta potential offers repulsive inter-particle interaction, preventing aggregation of the nanoparticles.⁵²

3.3.2. Entrapment efficiency. MTX–NPs showed appreciably higher entrapment efficiency (79.9 ± 2.4%), as compared to the CBA conjugated NPs (71.3 ± 1.8%) (Table 1). Dissolution and subsequent loss of surface adhered MTX from MTX–NPs upon addition of NPs in the medium may be ascribed to the divergence in entrapment efficiency. These outcomes are consistent with the results obtained in earlier investigations.⁵³

3.4. In vitro drug release profile

In vitro drug release at pH 7.4 and 5.4 on plain and conjugated NPs demonstrated a biphasic drug release pattern, suggesting an early burst, followed by a lag phase and a later apparent-zero order phase (Fig. 4C). MTX release of 20.26 ± 1.58% and 28.9 ± 2.45% was attained from CBA–MTX–NPs and MTX–NPs, respectively, in the PBS (pH 7.4). On the other hand, 21.4 ± 1.03% and 33.9 ± 1.15% of MTX were found to be released from CBA–MTX–NPs and MTX–NPs, respectively, in the acidic media (pH 5.4), until the end of the 8^th hour (Fig. 4C). The most probable reason for this rapid release of MTX can be the release of drug adsorbed on the surface of the NPs. Furthermore, at pH 7.4, the cumulative MTX release from CBA–MTX–NPs and MTX–NPs was 73.51 ± 3.64% and 88.3 ± 4.85%, respectively, at the end of 144 hours. In another case, 95.60 ± 3.53 of MTX was found to be released from CBA–MTX–NPs at the end of 144 h. The extended release could be ascribed to the diffusion of drug molecules across the polymeric milieu of the NPs. However, a sizeable decrease in the cumulative release of MTX from their CBA conjugated formulations was observed, in comparison to their unconjugated counterparts at both pH values (pH 7.4 and pH 5.4). This may be ascribed to the structural integrity conferred by CBA, thus providing a diffuse double layer obstacle and offering a steric hindrance to the diffusion of the drug.^9,54 Furthermore, the accelerated degradation of PLGA and the lessening of the ionic interactions between the drug and PLGA at an acidic pH might account for faster drug release at the lower pH. Acidic pH catalyzes the cleavage of ester linkages in the polymer backbone and augments the erosion of the polymeric matrix.⁵⁵ As a result, PLGA NPs could be anticipated to afford a pH-stimulating release of the entrapped MTX in the acidic milieu of the tumor and might facilitate ligand mediated transcytosis as well.

3.5. Ex vivo studies

3.5.1. Cytotoxicity and cell uptake studies. The cytotoxic response in terms of the percentage growth inhibition of free MTX, MTX–NPs and CBA–MTX–NPs was evaluated by SRB assay against C-6 glioma cells. The percent cellular inhibition response against all formulations was found to be concentration dependent. None of the formulations showed any effect on the survival of cells when the MTX equivalent concentration was below 1 μM, and in all these cases, the cell viabilities were found to be well above the 90% survival rate (Fig. 5A–C).

Fig. 5 Cell cytotoxicity of plain MTX, MTX–NPs, CBA–MTX–NPs and CBA + CBA–MTX–NPs (A) after 24 h, (B) after 48 h and (C) after 72 h. (D) Cellular uptake efficiency of plain FITC, FITC labeled MTX–NPs, FITC labeled CBA–MTX–NPs and CBA + FITC labeled CBA–MTX–NPs in C-6 glioma cells. Each data point is represented as mean ± SD (n = 4).

Experiments conducted on xenograft models undoubtedly exemplify a dose dependent cytotoxic response that is a decrease in the percent cell survival, with an increase in the concentration of the drug. Nearly 100% of the cells were killed when the concentration of MTX was 100 μM for all the formulations. The cytotoxic action of the drug probably depends on the adequate access of drug in the cell and not merely on its presence in the adjacent milieu of the cell over the incubation period (72 h). In the concentration range (1–100 μM) of MTX, the cytotoxic effect was sorted as CBA–MTX–NPs > MTX–NPs > CBA + CBA–MTX–NPs > MTX (Fig. 5A–C).

Maximum intracellular entry of CBA–MTX–NPs was conferred upon CBA conjugation, via adsorption mediated endocytosis. Consequently, the extended release of MTX leads to a considerably higher cytotoxic action, compared to free MTX or MTX–NPs. The cytotoxic effects via MTX–NPs were measured to be less than the CBA appended NPs, possibly due to their entry in the absence of any active transport mechanism, and were mediated merely by the passive diffusion mechanism. Furthermore, the nanometric size of the NPs may lead to the enhanced permeation and retention (EPR) effect, and the coating of CBA enhanced the adsorption, as well as cell permeability, due to changes in the cell membrane fluidity. Besides, the percent cellular inhibition was found to be significantly less when cells were incubated with CBA–MTX–NPs + CBA (1 mg ml⁻¹). This may be credited to the competitive inhibition of CBA functionalized NPs with free CBA. Free CBA was preferentially up taken by the cells, and therefore, little entry of CBA–MTX–NPs was observed. The results are in line with previous outcomes reported by Storm et al., 2002.⁵⁶

3.5.2. Cellular uptake study. Imperatively, CBA–MTX–NPs facilitate a higher cytotoxic response in C-6 cells; in vivo, the ligand anchored formulation would promptly experience transcytosis, therefore evading the possibility of a marked increase in the percent cellular uptake effect on C-6 cells, as compared to the unconjugated formulation and fluorescence marker alone.

The efficacy of cellular uptake as a function of CBA conjugation and incubation time was determined using flow cytometry. At two hours post incubation of plain FITC, FITC labeled MTX–NPs, FITC labeled CBA–MTX–NPs, and free CBA + FITC labeled CBA–MTX–NPs, the percentage of fluorescence in C-6 glioma cells was found to be 9.8 ± 2.4%, 14.1 ± 3.1%, 33.6 ± 3.3%, and 11.7 ± 3.2%, respectively (Fig. 5D). Fluorescence intensity was augmented with time (p ≤ 0.05), and was merely 32.7 ± 2.6%, 51.5 ± 3.2%, 78.6 ± 3.8%, and 39.6 ± 3.3%, respectively, after 6 h. The higher uptake of the CBA conjugated formulation was possibly due to CBA residues tethered on the facade of NPs when compared to unconjugated NPs (p < 0.05). This foremost cellular localization may be credited to the adsorption mediated endocytosis mechanism facilitated by CBA. Furthermore, FITC alone and FITC labeled MTX–NPs illustrated fluorescence due to the diffusion/endocytosis/phagocytosis mediated non-specific adsorption on cell surfaces. In contrast, the incubation of free CBA along with FITC labeled CBA–MTX–NPs led to distinctly restricted entry into the tumor cell lines. This may be ascribed to the competitive inhibition of CBA tethered NPs with free CBA. Free CBA preferentially gained access into the cells, and thus, the modest entry of FITC labeled CBA–MTX–NPs was evaluated. The outcomes are in line with previous studies.⁵⁶

3.6. In vivo pharmacokinetic and biodistribution studies

Biodistribution studies were carried out to examine the efficacy of CBA conjugated NPs in the delivery of anticancer agents to target tumor tissues, while circumventing the BBB and also bypassing peripheral tissues (kidney, heart, liver, etc.). Following the injection of MTX entrapped in NPs, the initial maximum concentration attained was slightly lower than plain MTX (Fig. 6A).

Fig. 6 (A) Biodistribution of plain drug and formulations attained at various time intervals in different tissues (n = 6; p ≤ 0.05*, ^a). *Significant difference between free MTX vs. MTX–NPs and CBA–MTX–NPs, ^asignificant difference between MTX–NPs and CBA–MTX–NPs. (B) Serum concentration of MTX attained at various intervals (n = 6; p ≤ 0.05*, ^a). *Significant difference between free MTX vs. MTX–NPs and CBA–MTX–NPs, ^a significant difference between MTX–NPs and CBA–MTX–NPs. (C) Comparative hemolytic toxicity study (n = 6; p ≤ 0.05*, ^a). *Significant difference between free MTX vs. MTX–NPs and CBA–MTX–NPs, ^asignificant difference between MTX–NPs and CBA–MTX–NPs.

After 2 h of administration, the MTX contents, when injected as a plain solution into the kidney, liver, heart, spleen, and tumor tissues, were 31.81 ± 1.41 μg g⁻¹, 10.13 ± 0.97 μg g⁻¹, 5.3 ± 0.7 μg g⁻¹, 8.1 ± 0.87 μg g⁻¹, and 2.9 ± 0.4 μg g⁻¹, respectively (Fig. 6A). The information evidently advocates the utmost NPs uptake by the liver/spleen (organs rich in RES cells), and there is access to the kidney, which is the primary organ for its clearance. A nominal concentration of MTX was estimated in the brain; the BBB acts as a main obstacle for delivery and further significantly impedes the therapeutic effectiveness. Furthermore, the concentration of MTX declined rapidly in all the organs, being untraceable in the kidneys and brain, 24 h after administration. This denotes the rapid removal/elimination/metabolism of the drug from the body when administered in its original form.

On the contrary, after 24 h of formulation administration, the concentrations of MTX loaded in the MTX–NPs were estimated to be: 11.51 ± 1.09, 7.22 ± 0.97, 3.31 ± 0.91, 6.95 ± 0.1.25 and 51.7 ± 3.4 μg g⁻¹ in the liver, kidney, heart, spleen and tumor, respectively (Fig. 6A). In the case of CBA conjugated NPs formulations, 112.69 ± 6.41 μg g⁻¹ of MTX was found in tumor tissues, 9.19 ± 0.95 μg g⁻¹ in the liver, with a lesser amount in the heart and 5.1 ± 0.57 μg g⁻¹ in the kidneys, and 5.63 ± 0.49 μg g⁻¹ in the spleen after 24 h (Fig. 6A). A sufficient amount of MTX was detected in the liver and spleen following the administration of MTX as MTX–NPs, and may be ascribed to the uptake of polymeric particles by mononuclear phagocytic system. A slight access of MTX concentration in brain tissues was also observed. An improved residence time of MTX in the systemic circulation was also noted upon incorporation of MTX inside the NPs, which facilitated the redistribution process of the drug into various tissues. Nevertheless, the deficiency of a targeting entity in the case of MTX–NPs led to non-selective/non-specific distribution and thus, the effect was not prominent.

The amounts of MTX determined in the kidneys, liver, heart, spleen, and brain tissues were 6.03 ± 0.39 μg g⁻¹, 9.24 ± 0.76 μg g⁻¹, 0.94 ± 0.04 μg g⁻¹, 8.03 ± 1.01 μg g⁻¹ and 19.89 ± 1.16 μg g⁻¹, respectively, 2 h following the administration of the CBA–MTX–NPs (Fig. 6A). Importantly, after 2 h, the MTX concentration with CBA–MTX–NPs in the brain was increased by 39.26 and 2.17 fold, when compared to free MTX and MTX–NPs. The outcomes undoubtedly advocate the targeted delivery of MTX to the brain with CBA decorated NPs. This may be ascribed to CBA tethering, which makes it possible for NPs to pass across the BBB by adsorption mediated transcytosis.^27,28 The stratagem may also facilitate enlarging the therapeutic efficacy by providing the possibility to diminish the dose of bioactive moieties and drug incorporated delivery vehicles.

The circulation life time, plasma profile, as well as the therapeutic effectiveness of MTX were enormously altered after the entrapment of MTX inside the nanoparticles and upon conjugation of CBA. Plasma profiles of various MTX formulations after a single IV injection in Balb/c mice are demonstrated in Fig. 6B. The concentration of MTX in serum after the IV administration of free MTX was estimated to be 1.57 ± 0.052 μg ml⁻¹ after 30 min and 0.13 ± 0.039 μg ml⁻¹ after 24 h. This steep decrease might be due to the rapid elimination of MTX from the kidneys and the instantaneous distribution in various organs. Conversely, after the administration of the MTX loaded nanoparticles formulation, the concentration of MTX in serum was significantly low. The maximum concentration of MTX estimated in the serum after 12 h was calculated to be 1.11 ± 0.047 and 1.19 ± 0.087 μg ml⁻¹ for MTX–NPs and CBA–MTX–NPs, respectively, although, 0.27 ± 0.041 and 0.68 ± 0.71 μg ml⁻¹ of MTX were measured in the plasma until the end of 24 and 20 h, following the administration of CBA–MTX–NPs and MTX–NPs, respectively (Fig. 6B). The outcomes merely signify the long circulation characteristic of NPs, while the ligand tethered formulation has the extended residence time. The concentration of MTX in the serum was sustained to a greater extent in the case of CBA–MTX–NPs, than MTX–NPs, probably due to the double barrier effect of drug diffusion upon surface modification with CBA.⁵⁴ The information offered up to this point merely implies that CBA–MTX–NPs have noticeably enhanced bioavailability and apparently extended retention in systemic circulation, and the results could not be accomplished via pre-treatment of mice with MTX–NPs or plain MTX.

3.7. Hemolytic toxicity

Concentration at 0.1 μM (equivalent of MTX), free MTX exhibited 17.9 ± 0.6% hemolysis; however, MTX–NPs and CBA–MTX–NPs formulations displayed 11.7 ± 0.3%, and 6.1 ± 0.51% hemolysis, respectively (Fig. 6C). The toxicity of polymeric NPs was not as great, in comparison to free MTX, due to the incorporation of drugs in a biocompatible nanostructured milieu. However, the hemolytic toxicity of NPs with functionalities at the surface is a major limitation to the use of such systems and was enough to exclude its use as a vehicle for drug delivery. CBA anchored to the NPs surface appreciably diminished the hemolysis of the RBCs, certainly due to the inhibition of communication of RBCs with the anticancer moiety associated with the surface of MTX–NPs.

4. Conclusions

NPs are the biocompatible nano-carriers that have been expansively investigated for the delivery of therapeutics. This is the first report of its kind that offers insight on the use of cationic bovine serum albumin tethered biodegradable polymeric nanostructured carriers for brain tumors, exploiting adsorption mediated transcytosis to circumvent the highly lipophilic BBB. The present study discloses CBA functionalized polymeric nanoparticles as proficient vectors to carry large doses of anti-cancer drugs for sustained and targeted delivery. CBA tethered NPs incorporating MTX demonstrated cytotoxicity higher than that of plain MTX on C-6 glioma cells at all the concentrations tested. The formulation proficiently navigated huge doses of anti-cancer therapeutic agent across the BBB. The most promising therapeutic response and diminution in annoying side effects may be facilitated by the site specific delivery strategy. Nevertheless, meticulous exercises are required in order to attain any unambiguous generalization.

Ethical statement

All animal studies were conducted in accordance with current legislation of the institute on animal experiments, and protocol was approved by the ‘Institutional Animal Ethical Committee’ of Dr Hari Singh Gour University, Sagar (M.P. India). Animals were maintained in climatically controlled rooms and fed with standard rodent food pellet (Lipton India Ltd, Bombay) and water ad libitum. The study was carried out with the guidelines of the Council for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India.

Conflict of interest

There is no conflict of interest and disclosures associated with the manuscript.

Acknowledgements

The authors are grateful for a grant and fellowship provided by the AICTE, New Delhi and Council of Scientific and Industrial Research (CSIR, HRDG), New Delhi, India.

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