Int J App Pharm, Vol10, Issue 6, 2018, 234-241Original Article


CARMUSTINE LOADED LACTOFERRIN NANOPARTICLES DEMONSTRATES AN ENHANCED ANTIPROLIFERATIVE ACTIVITY AGAINST GLIOBLASTOMA IN VITRO

HARIKIRAN ATHMAKUR, ANAND KUMAR KONDAPI*

Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, India
Email: akondapi@gmail.com

Received: 18 Jun 2018, Revised and Accepted: 04 Oct 2018

ABSTRACT

Objective: Despite sophisticated treatment regimens, there is no significant improvement in the mortality rates of glioblastoma due to insufficient dosage delivery, reoccurrence of tumors, higher systemic toxicity, etc. Sincebrain endothelial cells and glioblastoma cells express lactoferrin receptors, a target-specific drug delivery vehicle was developed using lactoferrin itself as amatrix,into whichcarmustinewas loaded.The objective was to usecarmustine loadedlactoferrin nanoparticles(CLN)to achieve higher therapeutic efficacy and target specificity compared to free carmustine.

Methods: CLN were prepared using the Sol-oil method. Thenanoparticles preparedwere characterized for their size, shape, polydispersity, and stability using FESEM and DLS methods.Drug loading and drug releasing efficiencies werealso estimated. Further, cellular uptake of nanoparticles and their antiproliferative efficacy against glioblastoma cells wereevaluated.

Results: Characterization of CLN showed that theywerespherical with ≤ 41 nm diameter and exhibitedhomogeneously dispersed stable distribution. Loading efficiency of carmustine in CLN was estimated to be 43±3.7%. Drug release from the nanoparticles was pH dependent with the maximum observed at pH 5. At physiological and gastric pH, drug release was lower, whereas maximum release was observed at endocytotic vesicular and around tumor extracellular pH.Confocal microscopic studies showed an active cellular uptake of nanoparticles. Results of antiproliferative analysis substantiated a higher antiproliferative effect for CLN compared to free carmustine.

Conclusion: The results of the study demonstrated that CLN serves as a vital tool, in designing an effective treatment strategy for targeted drug deliverytoglioblastoma.

Keywords: Lactoferrinnanoparticles,Carmustine,Glioblastoma, Drug delivery vehicle

© 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijap.2018v10i6.28004

INTRODUCTION

Brain and othercentral nervous system tumors(BCNST), are known to be one of the leading causes of cancerous deaths. According to the central brain tumorregistry of the United States (CBTRUS), in the US alone, every year on an average 15000 deaths occur due to BCNST, and approximately 80000 new cases are diagnosing yearly [1]. No known environmental risk factors other than ionizing radiation had identified for such higher incidence rates[2-4]. Amongst all the BCNST, glioblastoma is the most common and most aggressive malignant tumor with5 y post-diagnosis survival rates of less than 6% [1]. World health organization (WHO)grade IV classified, glioblastomaarises from malignantly transformed glial cells, and it diffusely invades other regions of the brain, making it highly lethal [5-7]. Its higher reoccurrence even after surgical resection adds to the complexity [8].

Current preferred treatment for glioblastoma is surgical resection of tumors, followed by radiotherapy with concurrent chemotherapy [9-13]. Despite these advanced treatments, there is no significant improvement reported in the overall survival rates of patients [8].These failures are mainly due to (a) reoccurrence of tumorsthat arise from surgically inaccessible infiltrating malignant cells [8];(b) emergence of resistance to radiotherapy and chemotherapy due to suboptimal dosage exposure for prolonged periods as a result of inefficient dosage delivery [14-17]; (c) higher systemic toxicity as a consequence of nonspecific localization of drugs [18-20]. These failures emphasize the need forthe development of efficientdrug delivery vehicles with significant drug localization in glioma cells.

In recent years, numerous efforts have been made to develop different drug delivery vehicles to overcome the above problems.Some of these are namelyliposomes, nanoshells, dendrimers, solid lipid nanoparticles, polymeric micelles, carbon nanotubes, polyglycolic acid (PGA) nanoparticles, polylactic acid (PLA) nanoparticles, poly(D,L-lactic-co-glycolides) acid (PLGA) nanoparticles, polyanhydride nanoparticles, polyorthoesters nanoparticles, polycyanoacrylate nanoparticles, polycaprolactone nanoparticles, chitosan nanoparticles, albumin nanoparticles,etc. [21, 22]. But, many of these drug delivery vehicles lack target specificity, making their scope limited. Further, the poorability of these vehicles in the transport of drugs across theblood-brain barrier significantly limits their application for delivery of drugs to the brain. Many strategies have developed to overcome the above limitations[22, 23].Among them, exploiting one of the brain’s natural transport systems, the receptor-mediatedendocytosis, is gaining much interest in delivering therapeutic drugs to the brain. Ligands commonly used for this purpose are folate, transferrin, lactoferrin, etc. These ligands are either coated or conjugated to the nanoparticles, to facilitate nanoparticles entry into the brain via receptor-mediated endocytosis [23, 24].

Lactoferrin is an 80 kDa protein, which is mainly found in milk and other secretory body fluids. It has numerous clinically significant physiological functions viz., anti-inflammation, host defense against infections, maintenance of iron homeostasis, etc.[25-28]. Sincebrain endothelial cells, and glioblastoma cells [29-33] express lactoferrin receptors and alsoits low endogenous levels in serum [34, 35], make lactoferrin more advantageous in using it for targeting to the brainas it avoids competition with endogenous ligands and also increases target specificity.Drug-loadednanoparticles were reported to possess asignificant advantage over drug conjugated nanoparticles regarding efficacy and drug release in the targeted cells [36].

In the context of these facts, biodegradable protein nanoparticles were developed using lactoferrin itself as a matrix, into which chemotherapeutic drug, carmustine was loaded, and thesenanoparticles were used for targeting brain tumorsin vitro. The objective was to exploit lactoferrin nanoparticles for a dual purpose, as a drug carrier, as well as a targeting ligand. Cell culture models were used to evaluate the efficiency of carmustine loaded lactoferrin nanoparticles in drug localization and cytotoxicity.

We hypothesize that carmustine loaded lactoferrinnanoparticles will be an effective treatment strategy for targeting brain tumors if it increases target specificity, enhance therapeutic efficacy, bioavailability,and stability, and also minimizes the systemic toxicity of the drug. This paper discusses the preparation of carmustine loaded lactoferrinnanoparticles, their optimal characteristic features which make them better drug delivery vehicles and further about their efficacy in treating brain tumors in general and more particularly glioblastomain vitro.

MATERIALS AND METHODS

Materials

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4′,6-diamidino-2-phenylindole (DAPI), rhodamine123 were procured from Sigma–Aldrich (St. Louis, USA),lactoferrinwas obtainedfrom Naturade LLC (Irvine, USA), andolive oil from Nicola pantaleo (Fasano, Italy). Carmustine was of pharmaceutical grade(Emcure pharmaceuticals, Pune, India). Minimum essential media, non-essentialamino acids, sodium pyruvate, Dulbecco's modified eagle medium, fetalbovine serum were bought from Thermo fisher scientific (Waltham, USA). C6glioma, SK-N-SH cell lines were acquired from National centre for cell science(Pune, India). Rest of the materials were of either analytical or molecular biological grade.

Preparation of carmustine loaded lactoferrin, blank lactoferrin and rhodamine loaded lactoferrin nanoparticles

Nanoparticleswere prepared as described byKrishna ADet al. (2009)[36]. Briefly, 1 ml of coldphosphate-buffered saline (PBS) pH-7.4 containing 50 mg of dissolved lactoferrin was gently mixed with 20 mg of carmustine dissolved in dimethyl sulfoxide (DMSO). The mixture was incubated at 4°C for 30 min. After incubation, the mixture was slowly added to 30 ml of cold olive oil and was gently dispersed by vortexing, followed by sonication using ultrasonic homogenizer at 4°C. Immediately the resulting mixture was snap frozen by keeping it in the liquid nitrogen for 10 min. After thawing the mixture at 4 °C, it was centrifuged at 8000 g for 15 min at 4 °C. Thesupernatant was discarded, and the pellet was washed thrice with diethyl ether. Following air drying, the pellet was dispersed in cold PBS (pH-7.4) by sonication and was stored at 4°C until use. For fluorescent studies, rhodamine loaded lactoferrinnanoparticles were prepared similarly, but instead ofcarmustine,rhodaminewas used. Similarly, blank lactoferrinnanoparticles were also prepared, but without the use of drug or dye.

Characterization of nanoparticles by field emission scanning electron microscope (FE-SEM)

Size and morphology of lactoferrinnanoparticles were characterized by FE-SEM (Field electron and ion, Hillsboro, USA). Freshly prepared lactoferrin nanoparticles were coated on a clean glass slide and were dried overnight in a dust-free chamber. Samples were then coated with gold and were viewed under the electron microscope. For image capturing and data analysis,the manufacturer’s standard operative procedures were followed.

Characterization of nanoparticles bydynamic light scattering (DLS)

Zeta potential, hydrodynamic diameter, andpolydispersityindex (PDI) of lactoferrinnanoparticles in suspension form were analyzed by dynamic light scattering method using SZ-100 Nanoparticaanalyzer system equipped with a diode-pumped solid-state laser having a wavelength of 532 nm (Horiba scientific, Irvine, USA). Particle analysis and data acquisition were carried out according to the manufacturer’s instructions.

Evaluation of loading efficiency

Carmustine loaded lactoferrinnanoparticleswere suspended in 1 ml of PBS of pH-5 and were kept under gentle rocking at 4°C for 30 min for the release of the drug from the nanoparticles (n=3). 30% silver nitrate was added to precipitate the protein out of the solution. The resulting solution was centrifuged at 15 000 g for 15 min at 4°C. The obtainedsupernatantwas filtered and used for the drug estimation byhigh-performance liquid chromatography (HPLC) (Waters, Milford, USA)[37]. The supernatant was analyzed in triplicate. Different concentrations of carmustine solutions were also prepared and estimated by HPLC to develop a standard curve. Amount of carmustine loaded in the lactoferrin nanoparticles was determined using the developed standard curve.

Drug loading efficiency was calculated using the following formula.

whereDLoaded = amount of loaded drug; DTotal = amount of total drug used; DLost= amount of drug lost during preparation.

In vitro pH-dependent drug release assay

pH-dependent drug release assay was performed by quantifying drug released under different pH conditions [38].Pelleted nanoparticles equivalent to 200 μg of carmustine were suspended in PBS solutions of varying pH ranges (1-9) and were incubated for 4 h at 4°C on a rocker with moderate speed. After incubation, 30% silver nitrate was added to the PBS solutions to precipitate protein. The mobile phase was also added to extract the drug, followed by centrifugation at 15000 g for 15 min at 4°C. The obtained supernatant was filtered using a0.2-micron filter, and the amount of drug present in the supernatant was estimated using HPLC at 230 nm wavelength for carmustine. For quantification of unknown amounts of the drug in the samples, a standard curve was developed using known concentrations of the drug in the same incubation media and quantified by HPLC. Each sample was quantified in triplicate (n = 3).

Cellular uptake assay by confocal microscopy

SK-N-SH cells (seeding density of 2 × 10⁵ cells) were grown on glass coverslips in 12 well plates. Equivalent amounts of rhodamine loaded lactoferrin nanoparticles were added to the wells and were incubated for different time points (0.5 h, 1 h, 2 h, 4 h, and 8 h). Untreated cells were kept ascontrol. After specified time points, cells were washed thrice with PBS buffer (pH-7.4) and were fixed with 4% paraformaldehyde for 10 min. After subsequent washings with PBS buffer, cells were counterstained with DAPI, and the coverslips were mounted on a glass slide. Cells were viewed under the confocal microscope(Leica, Buffalo grove, USA) for analyzing the amount of uptake of nanoparticles, by utilizing the intrinsic fluorescence of rhodamine123 (excitation and emission maxima are 511 nm and 534 nm respectively)[39].

Evaluation of the antiproliferative activity of carmustine loaded lactoferrin nanoparticles

The antiproliferative assay was performed using the MTT method [40]. Briefly, 50000 C6 glioma cells were seeded in every well of the 96 well plate and were incubated in the carbon dioxide incubator at 37°C for 12 h. After incubation, media was replaced with fresh media containing increasing concentrations of either soluble carmustine or its equivalent carmustine loaded nanoparticles. Similar treatment was given with blank lactoferrinnanoparticles. Control cells were also kept, without the addition of neither soluble drug nor the nanoparticles. Cells were incubated in the 37°C carbon dioxide incubator for 24 h. After incubation, media was discarded, and cells were washed twice. Fresh media containing 10%, 5 mg/ml MTT, was added to the cells followed by incubation for 8 h in a carbon dioxide incubator at 37°C. During incubation, cells that survived after the treatment convert yellow tetrazolium salt into insoluble formazan crystals. MTT containing media was discarded, and insoluble formazan crystals were dissolved by the addition of DMSO. The intensity of the developed color was measured using multiplate reader-Infinite 200 (Tecan,Mannedorf, Switzerland) at 595 nm. Percentage of inhibition (PI) was calculated according to the following formula.

where, PI = percentage of inhibition; ODControl = absorbance at 595 nm for control cells; ODTreated = absorbance at 595 nm for treated cells.

After plotting the graph, half maximal inhibitory concentration (IC50 value) was calculated from it.

Statistics

All the experiments were performed a minimum of three times individually. Data were presented as mean±standard deviation. Amounts of drug released at various pH conditions were statistically analyzedby one-way ANOVA using the Student-Newman-Keulsmethod. Antiproliferativeactivities of free carmustine and carmustine loaded lactoferrin nanoparticles were statistically compared by Student t-test. P<0.05 was regarded as statistically significant.

RESULTS

Characterization of nanoparticles by FESEM

Blank lactoferrinnanoparticles and carmustine loaded lactoferrinnanoparticles were prepared as described in materials and methods. The prepared nanoparticles were characterized by FESEM to obtain information relating to their size and morphology. The FESEM analysis revealed that their sizes were in the range of 13-22 nm, with an average size of 17.5±3.06 nm (mean±SD) for blank lactoferrin nanoparticles (fig. 1a) and in the range of 32-41 nm, with an average size of 36.5±3.90 nm(mean±SD) for carmustine loaded lactoferrin nanoparticles (fig. 1b). It is apparent that lactoferrinnanoparticles become more than double in their average size after loading of the drug. Further, FESEM analysis revealed that the nanoparticles were homogenous in their sizes and were spherical in their shapes.

(A) (B)

Fig.1: FESEM analysis of (A) blank lactoferrinnanoparticles, (B) carmustine loaded lactoferrinnanoparticles. Above image was representative of a quadruplicate experiment (n = 4)

Characterization of nanoparticles by DLS

Zeta potential values of blank lactoferrinnanoparticles and carmustine loaded lactoferrinnanoparticles were-14.9±3.87 mV (mean±SD) (fig. 2a) and-24.6±5.94 mV (mean±SD) (fig. 2b) respectively. These zeta potential values indicate that carmustine loaded lactoferrinnanoparticles were under colloidal stability in nature, andblank lactoferrinnanoparticles were under moderately colloidal stability in nature. Hydrodynamic sizes of nanoparticles were also investigated using DLS analysis (fig. 2c and fig. 2d). Since DLS measures the hydrodynamic diameter of the particles, whereas FESEM measures size in the dry state, nanoparticles sizes were little larger in DLS compared to FESEM analysis. PDI for blank lactoferrinnanoparticles was 0.264±0.03 and for carmustine loaded lactoferrinnanoparticles was 0.338±0.05. These PDI values confirm that these nanoparticles had a homogeneously dispersed size distribution.

Fig.2:DLSanalysis: Zeta potential measurements of (A) blank lactoferrinnanoparticles, (B) carmustine loaded lactoferrinnanoparticles. Hydrodynamic diameter measurements of (C) blank lactoferrinnanoparticles, (D) carmustine loaded lactoferrinnanoparticles.The experiment was conducted in triplicates (n = 3)

Estimation of drug loading efficiency of carmustine loaded lactoferrin nanoparticles

Significantly higher drug loading efficiency was achieved in the nanoparticles by using theSol-oil method. Carmustine solutions of different concentrations were prepared and estimated by HPLC, and the standard graph was developed for calculating the amount of encapsulated carmustine present in the lactoferrinnanoparticles (fig. 3b). Carmustineloaded lactoferrinnanoformulations were treated as described in the materials and methods,the released drug was estimated by HPLC (fig. 3a) and correlated with the standard graph. Then the loading efficiencies were calculated using the formula mentioned in the materials and methods. Loading efficiency of carmustine in thecarmustine loaded lactoferrinnanoparticles was found to be 43±3.7% (n=3).

Fig.3: (A) HPLCanalysis of carmustine at 230 nm wavelength. (B) Quantification of carmustine by HPLC; data were represented asmean±SD(n = 3)

In vitro pH-dependent drug release assay

pH-dependent release assay of carmustine loaded lactoferrin nanoparticles were carried out at various pH ranges (1–9) (fig. 4). The maximum amount of drug was released at pH 5, which was followed by pH 6. At all other pH conditions, the release was comparatively low. At physiological pH (pH 7.2 to 7.4) and gastric pH (pH 1 to 2.5), drug release was less than 20% and whereas maximum release was observed at endocytotic vesicular pH (pH 5) and around tumor extracellular pH (pH 5.85-7.35) [41], from the nanoparticles.

Fig.4: pH-dependentrelease assay of carmustine loaded lactoferrinnanoparticles. Car-lacto represents carmustine loaded lactoferrin nanoparticle. Averages and standard deviations from three experiments (n = 3) were shown as mean±SD. **P<0.01 byone-way ANOVA using Student-Newman-Keuls method

Cellular uptake assay by confocal microscopy

Rhodamine123 loaded lactoferrin nanoparticles were incubated with the cells for different time points to confirm the cellular uptake of nanoparticles. Up to 1 h of incubation, rhodamine was not visible, but after 2 h, it was noticed that its level had increased with the increment of time. By the end of 8 h, cells were localized entirely with rhodamine123. Cells, which were not exposed to rhodamine123 loaded nanoparticles, were taken as the control (fig. 5). Time course experiment showed that there was a gradual increase of rhodamine in the cells with the time, which confirmed the cellular uptake of nanoparticles and their rise was gradual and proportional to the time. This result also confirmed the longer retention of lactoferrin nanoparticles within the cells, which provides a longer time for chemotherapeutic drugs to conferthe antiproliferative effect.

Fig.5: Cellular uptake of rhodamine123 loaded lactoferrin nanoparticles.Time course experiment showed the uptake of nanoparticles into the cells, andthere was a gradual increase in the uptake of nanoparticles with the increment of time. A, B, C, D, E, F represent control, 0.5 h, 1h, 2h, 4h, and 8h time points respectively. In each big square, the upper left square represents the rhodamine 123 (red), the upper right square represents transmission image, the lower left square represents DAPI (blue), and the lower right square represents merger image. Total number of independent experimentation, n = 3

Evaluation of the antiproliferative activity of carmustine loaded lactoferrin nanoparticles

Antiproliferative activity of carmustine loaded lactoferrin nanoparticles were compared with the antiproliferative activity of the free drug (carmustine) after 24 h of treatment with free drug and drug-loaded nanoparticles. The results clearlyshowed that carmustine loaded lactoferrin nanoparticles had a higher antiproliferative effectcompared to free carmustine at all the experimental concentrations (fig. 6a). And there wasa reduction of 3.29 times in the IC50 value with the treatment of carmustine loaded lactoferrinnanoparticles compared to free carmustine treatment.IC50 values of free carmustine and carmustine loaded lactoferrinnanoparticles were 43.22 μg/ml and 12.76μg/ml respectively. Blank lactoferrinnanoparticles (delivery vehicle) didn’tshow any significant antiproliferative activity at all the experimental concentrations (fig. 6b).

(A) (B)

Fig.6: Dose-dependentantiproliferative activities of (A) free carmustineandcarmustine loaded lactoferrin nanoparticles and (B) free lactoferrin nanoparticles after 24 h of treatment. Car, car-lacto, andlacto-nanorepresent thetreatment of carmustine drug,carmustineloaded lactoferrin nanoparticles and blank lactoferrin nanoparticles respectively. Data were represented as mean±SD(n = 3), **P<0.01 by student t-test

DISCUSSION

Current advanced treatments for glioblastoma remain not so effective since there is no significant improvement in the survival of the patients. The purpose of this study was to develop an effective target-specific drug delivery vehicle with reduced systemic toxicity and increased therapeutic efficacy againstglioblastoma. As it is known, lactoferrin receptors are expressed on brain endothelial cells, and glioblastoma cells, a target-specific drug delivery vehicle was developed, using lactoferrin itself as a matrix, into which chemotherapeutic drug, carmustine was loaded. In the present study, carmustine loaded lactoferrinnanoparticles were prepared and characterized their features such as size, shape, polydispersity, stability, drug loading efficiency, drug releasing efficiency, cellular uptake ability, etc., and further evaluated their efficacy in treating glioblastoma.

For decades, carmustine was a drug of choice for treating glioblastoma[42-44]. Due to its dose-limiting side effects such as bone marrow suppression [45] and non-dose dependent pulmonary fibrosis [46], its usage was limited. To reduce systemic toxicity, intracranial polymer implants (gliadel wafers) impregnated with carmustine, have been using clinically since 1996 [19]. But these gliadel waferswere found to be not successful as they do not show effective therapeutic efficacy due to many limitations such as lower penetration, inability in preventing short distant tumor recurrence, lack of synergetic action in combination with radiotherapy and other chemotherapeutic drugs, practical difficulties in prescribing regular dosage schedules as it requires regular intracranial surgeries [47, 19]. Further, complications are reported in the use ofgliadel wafers due to severe adverse effects such as healing abnormalities [48], craniotomy infections[49], seizures [11], oedema [11, 50], neurological decline [50], intracranial hypertension [10], cerebrospinal fluid leaks [10], tumor bed cyst formation [51], pericavity necrosis [52] etc.These limitations and adverse effects emphasize the need for a better drug delivery vehicle for efficient treatment of glioblastoma.

In the present study, carmustine loaded lactoferrinnanoparticles were prepared using the Sol-oil method. This method is simple, less time consuming and doesn’t involve any chemical modifications either to the drug or protein unlike other methods such as protein coacervation method [53]. Further, lactoferrin conformation remains in the native state. As the nanoparticles are prepared using lactoferrin, a natural protein present abundantly in milk and other secretory body fluids, these nanoparticles are safer to use even at high dosages. Besides, significantly higher drug loading efficiency was also achieved in the nanoparticles by using this method. Drug loading was indeed higher compared to commercially available carmustine implants, which have only 3.85% drug loading capacity [11]. The reported maximum drug loading capacity that can be achieved in the biodegradable polymers was 28% [54], which was less than that observedin carmustine loaded lactoferrin nanoparticles(43±3.7%). Drug loading efficiency of a drug delivery vehicle can influence its therapeutic index. Higher the achieved drug loading efficiency, higher will be the therapeutic index. With the increased therapeutic index there will be an enhancedantitumor effect and reduced toxicity [55].

Using Sol-oil method, nanoparticles of sizes ≤ 41 nm were successfully developed, which was confirmed by FESEM analysis. FESEM studies also showed that blank lactoferrin nanoparticles of 13-22 nm size became enlarged to 32-41 nm size after successful loading of carmustine drug. It is an advantage to have smaller sized nanoparticlesbecause several studies had consistently shown that smaller sized nanoparticles were capable of escaping from thereticuloendothelial system, thereby evaded rapid clearance from systemic circulation and had longer circulation time and stability in the blood [56-59]. Several other studies also had shown, an inverse correlation between nanoparticle size’s and blood-brain barrier penetration [60-62]. It indicates that particles, which are smaller in size can cross theblood-brain barrier more efficiently than particles which are larger. Thus, the smaller size of these nanoparticles can increase their circulatory half-life and also make them more efficient in crossing the blood-brain barrier.

The measured zeta potential values indicate that these nanoparticles were stable. Carmustine loaded lactoferrinnanoparticles were under colloidal stability range, whereas blank lactoferrinnanoparticleswere under moderately colloidal stability range.Measured nanoparticle sizes were found to be little larger in DLS compared to FESEM analysis. Since, DLS measures the hydrodynamic diameter of the particle, which includes not only the particle but also the ionic and solvent layers associated with the particle in the solution, the particle sizes will be larger in DLS compared to FESEM, which measures size in the dry state [63, 64]. Polydispersityindex values of 0.264±0.03 and 0.338±0.05 for blank lactoferrinnanoparticles and carmustine loaded lactoferrinnanoparticles respectively indicate that these nanoparticles possess a homogenous population withnarrowsize distribution.

The release of the drug from the nanoparticles wasfound to be pH dependent. It was observed that at physiological and gastric pH, drug release from the nanoparticles was very minimum, whereas maximum drug release was observed at the endocytotic vesicular pH (pH 5) and around tumor extracellular pH (pH 5.85-7.35) [41], which indicates that these nanoparticles can have low loss of the drug during systemic circulation, thereby exhibit reduced systemic toxicity. And they also show more specificity in the drug release, mainly in the endocytotic vesicles, which are involved in receptor-mediated endocytosis and around tumor environment, which have reduced pH as a consequence of higher anaerobic respiration of cancerous cells [65, 66]. pH-dependent drug release is an added advantage, which makesthesenanoparticles optimal drug delivery vehicles with reduced systemic toxicity and increased tumor specificity.

As carmustine drug was nonfluorescent, cellular uptake of nanoparticles was tested by loading fluorescent dye, rhodamine123 into the lactoferrin nanoparticles. Time course study using confocal microscopy hadshown that there was a gradual increase of rhodamine in the cells with the increment of time, which confirmed the active cellular uptake of lactoferrinnanoparticles. Earlier it was reported that the mechanism of uptake of lactoferrinnanoparticles into the cells was through receptor-mediated endocytosis [36, 39]. As the brain endothelial cells and glioblastoma cells express lactoferrin receptors [29-33], a similar mechanism could be operative, during the transport of carmustine loaded lactoferrinnanoparticles across the blood-brain barrier and also at the entry into the tumor cells.

Comparative study of the antiproliferative effect of free carmustine and carmustine loaded lactoferrinnanoparticles, had validated that carmustine loaded lactoferrinnanoparticles had a higher therapeutic efficacy than free carmustine. Previously, carmustine encapsulated liposomes, and carmustine-magnetic nanoparticles showed 50% inhibition at around 467 μM and 100 μM concentrations of carmustine respectively [67, 68], whereas carmustine loaded lactoferrin nanoparticlesshowed 50% inhibition at59.6 μM of carmustine, which was significantly lower and further substantiates the higher therapeutic efficacy of carmustine loaded lactoferrinnanoparticles.These results were also consistent with the earlier reports [39, 38], where lactoferrin nanoparticles had used as drug delivery vehicles. Higher uptake of nanoparticles, sustained drug release from the nanoparticles and the longer retention of the drug inside the cells might have contributed to the increased therapeutic efficacy of carmustine loaded lactoferrinnanoparticles against C6 glioma cells compared to the free carmustine.

Current state of the art, drug delivery vehicles for the treatment of glioblastoma includes solid lipid nanoparticles, nanostructured lipid carriers, liposomes, polymeric nanoparticles, micelles, magnetic nanoparticles, gold nanoshells, carbon nanotubes, etc. These drug delivery vehicles are failing to be an effective therapeutics due to one or more of the crucial issues viz., lower encapsulation efficiency, higher toxicity, lower stability, rapid clearance from the blood, lower biodegradability, lack of specificity, lower therapeutic indices, higher manufacturing costs, etc. [69-71, 21]. But,as may be seen from the results of the present study,carmustine loaded lactoferrinnanoparticles are showing promising results, which may overcome these challenges.

Thus, carmustine loaded lactoferrinnanoparticles serve as potential drug delivery vehicles in treating glioblastoma effectivelyin vitro. Further studies are required to establish in vivo efficacy.

CONCLUSION

Carmustine loaded lactoferrinnanoparticles, with ≤ 41 nm size were successfully developed, using lactoferrin as a single matrix. These nanoparticles werespherical with homogeneous distribution, enhancedstability, and higher drug loading efficiency. The release of the drug from nanoparticles was pH dependent, which adds additional advantage to this target specific drug delivery vehicle. Further, active cellular uptake of nanoparticles with a significant antiproliferative effect in cell culture models substantiatedcarmustine loaded nanoparticles as an effective drug delivery vehicle in treating glioblastoma.

Further in vivo efficacy and toxicological studies using carmustine loaded lactoferrinnanoparticles would provide an opportunity for the development of an effective treatment strategy against glioblastoma without any systemic toxicity.

ACKNOWLEDGEMENT

Authors are thankful to the Indian Council of Medical Research and Department of Science and Technology, India for providing the grant for this work. Authors are grateful to the University Grants Commission, India and the Department of Biotechnology-Centre for Research and Education in Biology and Biotechnology program of the University of Hyderabad, India for providing the infrastructure for this work. Authors are also thankful to the Council of Scientific and Industrial Research, India for providing doctoral fellowship to Harikiran.

AUTHORS CONTRIBUTIONS

Harikiran performed all experiments, compiled data, and drafted manuscript. AnandKumar planned experiments, analyzed results and edited manuscript. All the authors have approved the final article.

CONFLICT OF INTERESTS

Declared none

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