INTRODUCTION:

There has been a rapid increase in the occurrence of malignant melanoma in the industrialized countries for the last 30 years¹. As per the estimates of American Cancer Society, around 76,380 new melanoma cases will be diagnosed in 2016, with a predictable mortality of 10,130 people. It’s one of the most common cancers in young adults (especially young women). Despite the fact that there is a greater understanding of the risk factors, along with the genetic and epigenetic causes of melanoma, the mortality rate from melanoma is quite predominant compared to any other types of cancer².

Risk factors of melanoma take account of gene polymorphism, skin type, many moles on body or unusual moles, family history, a previous melanoma occurrence, and sensitivity towards sun, weakened immune system, and excessive ultraviolet (UV) exposure. Common treatments for malignant melanoma involve surgical procedures, chemotherapy and radiotherapy. Despite these stated treatments, at least one third of patients with early-stage melanoma develop metastasis, and the prognosis of metastatic melanoma remains dismal. Alarmingly, studies show that the melanoma patients have a median survival of approximately 6–8 months only. Moreover, it has been grimly observed that less than 5% of patients survive for a maximum of five years. Various chemotherapeutic drugs have been ineffective against melanoma cells as these cells become resistance to apoptotic pathways and leads to poor clinical outcome³. Lenalidomide is recently FDA approved standard drug used broadly for solid tumors ^{4, 5}.

Nanoparticle based treatments have offered promising clinical outcomes due to their tunable pharmacokinetic properties like prolonged half life, larger surface to volume ratio, enhanced drug solubility, and the likelihood for controlled release⁶. In particular, nanoparticles can be exploited for the targeted delivery of anticancer drugs at the tumor site. Target-specific nanoparticles render a new dimension to the standard conventional chemotherapy. A myriad of biomaterials including lipids, polymers, chatoyant and proteins has been used as matrix for nanoparticles formulation. Amongst which proteins, have garnered a significant interest as biomaterials due to three major associated inherent advantages. Firstly, proteins are biocompatible and show biodegradability⁷. Although some proteins have minor immunogenicity⁸, no substance-related toxicity was found as in case of engineered nanoparticles such as carbon annotates, dendrimers and quantum dots⁹. Secondly, drug binding, imaging or targeting becomes much simpler in case of proteins as it offers numerous moieties for modification. Thirdly, due to the existence of charged groups in proteins they can be a better matrix in which drugs can be physically entrapped. There are many proteins viz. albumin, gelatin, transferring, and elastin ^10-13 which have been used as nanocarriers for several drugs. Nanoparticles can target tumor tissues both actively and passively. The passive targeting is by enhanced permeability and retention effect whereas; active targeting is achieved by functionalization with ligands.

Lactoferrin (Lf), belong to transferrin family with a molecular weight of 80 KDa, is an iron carrying basic protein with numerous physiological roles. Due to its unique antimicrobial, antifungal, anticancer and anti-inflammatory properties, lactoferrin seems to have great potential in clinical sector. It has been used as a ligand for targeting several nanocarriers ¹⁴ but none of them utilized Lf as a matrix.

The present study is aimed at developing and optimizing Lf nanoparticles (LfNP) to enhance the therapeutic efficacy of Lnd in ovarian (SK-OV-3) in vitro. Lnd loaded Lf nanoparticles (Lnd-LfNPs) were prepared and characterized by TEM, SEM and DLS. Their loading efficiency, stability and in vitro drug release profile were studied. In addition to this, particles uptake mechanism, comparative cytotoxicity of free Lnd and Lnd-LfNPs were investigated in SK-OV-3 cells.

MATERIALS AND METHODS:

Materials:

Lactoferrin and olive oil used for nanoparticles preparation were purchased from Symbiotic (USA) and Leonardo (Italy), respectively. Lnd and MTT ((3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) was purchased from sigma Aldrich. All other chemicals and reagents were of analytical grade.

Preparation of Lnd-LfNPs:

Lnd-LfNPs were prepared by sol-oil method as described earlier¹⁴. Briefly, Lnd was mixed with Lf in a ratio of 1:2 (w/w) and incubated for 30 min on ice and then slowly added to 15 ml of olive oil at 4 °C with continuous dispersion by vortexing, followed by sonication at 4 °C. The resulting mixture was snap frozen and then thawed over ice. The particles were pelleted by centrifugation at 30,000 × g for 20 min and the pellet was extensively washed thrice with diethyl ether followed by PBS to remove excess oil and free Lnd. Finally the pellet was dispersed in phosphate buffered saline (PBS) and stored at 4 ºC or lyophilized as per the experimental constraint. For fluorescent labeling, blank lactoferrin nanoparticles (LfNPs) were prepared by similar procedure as stated above without the addition of Lnd and then was incubated with 50μl of 20% Rhodamine123 for overnight at 4 ºC to make Rhodamine123 tagged lactoferrin

nanoparticles (LfNPs-Rh123).

Physico-chemical characterization of nanoparticles:

Transmission Electron Microscopy (TEM) and Scanning electron microscope (SEM) analysis was carried out to study the morphology and size of nanoparticles. For TEM analysis, LfNPs were then placed on carbon coated copper 300 mesh grids, air dried and stained using 1% aqueous solution of uranyl acetate for 1 min. The samples were examined using Transmission Electron Microscope (JEM-2100, M/S Jeol Limited, Tachikawa, Tokyo, Japan). SEM analysis was carried out by coating nanoparticles onto the glass slides. Afterward slides were sputtered by silver paint. Particles were scanned in and data was analyzed according to manufacturer's instruction. The hydrodynamic diameter, poly dispersity index (PDI) and zeta potential were analyzed by nanoparticles analyzer system (Horiba Scientific, USA). For the in vitro storage stability studies, Lnd-LfNPs were prepared as described earlier, and stored in PBS at 4 ºC. At regular time intervals, aliquots were withdrawn and analyzed for their hydrodynamic diameter using a nanoparticle analyzer system (Horiba Scientific, USA) over a period of 5 weeks.

Determination of drug encapsulation efficiency and drug loading content:

The encapsulation efficiency was determined by re-suspending 0.5 mg of Lnd-LfNPs in 1ml PBS (0.01 M, pH 5.0) and incubated for 4 h at 37 ºC on a rocker. Equal volume of acetonitrile was then added to precipitate the protein, followed by centrifugation at 30,000 × g for 15 min. The supernatant was collected and filtered through a 0.2 micron syringe filter and quantified using UV-Vis spectrophotometer (JASCO) at 315 nm. All experiments were performed in triplicates. Encapsulation efficiency and drug loading content was calculated by following formula

E(%) =[Cd – C/Cd] x 100

In vitro pH release studies:

Lnd-LfNPs (0.5 mg) were re-suspended in 1 ml of buffers (ranging from pH 1.0 to 9.0) and incubated for 4 h on rocker at 37 ºC. Individual samples were collected by centrifugation at 30,000 x g for 15 min. Lnd released from the nanoparticles was quantified using UV–Vis spectrophotometer at 315 nm. All assays were done in triplicates.

Cell culture:

Mouse melanoma (B16F10) cell line were maintained in a Dulbecco’s modified Eagle’s growth medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 5 μg ml^-1 penicillin, 6 μg ml^-1 streptomycin, and 10 μg ml^-1 kanamycin. All cells were grown and maintained in 5% CO2, 95% humidity at 37 °C.

Toxicity studies:

The cytotoxicity of Lnd-LfNPs in B16F10 cells, was measured by MTT assay and compared with free Lnd. Briefly, cells were seeded in a 96-well plate at a density of 5000 cells per well and incubated at 37 ºC with 5 % CO₂ and 95 % humidity. After obtaining the desired confluency, cells were incubated with increasing concentrations of either free Lnd or Lnd-LfNPs (ranging from 5 to 50 μg ml-1). Cells were then incubated for another 24 h and the % cell inhibition was calculated using the following formula.

Test absorption value

% Cell Viability =---------------------------- X 100

Control absorption value

In order to determine the toxicity of the drug carrier (blank LfNPs without Lnd), cells were cultured in 12-well plate and incubated with increasing concentrations (5 to 1000 μg ml^-1) of blank LfNPs for 48 h. The cells were scraped, washed and fixed in 70% ethanol followed with addition of presidium iodide (PI) at a final concentration of 0.5 μg ml^-1. The samples were analyzed using flow cytometry as described above.

RESULTS AND DISCUSSION:

Physico-chemical characterization of nanoparticles

The Lnd-LfNPs were prepared by our previously reported patented protocol sol-oil chemistry¹³. The structure and morphology of the nanoparticles were characterized by TEM and SEM suggests that particles are spherical in shape with the size around 90 nm (Figure 1A and B).

(A)

(B)

Figure 1A and 1B: TEM and SEM images of nanoparticles

As expected the hydrodynamic diameter of the particles, as determined by DLS, was found to be 140 ± 10 nm (Figure 1C). The higher size obtained from DLS is due to the hydration shell present on the surface of the nanoparticles in DLS measurements, which was absent when analyzed in a dry state using TEM or SEM. The poly dispersity index (PDI) value and zeta potential of the particles was found to be 0.332 ± 0.1 and -2.5 ± 1 mV respectively, an indicative of a narrow size distribution with near neutral surface charge on the nanoparticles suggesting a stable dispersion of nanoparticles in aqueous media. Particles size and surface charge play a major role in cell uptake. The small size of the particles and a near neutral surface charge (ζ potential) of the LfNPs may help in evading their phagocytic uptake by macrophages, resulting in prolonged in vivo circulation and improved pharmacokinetics of Lnd¹⁶. The encapsulation efficiency (EE) of Lnd in LfNPs was found to be 64 ± 3.60% with a drug loading content (DLC) of 31.21 ± 2.5%. The high encapsulation efficiency and DLC, suggest significantly high Lnd encapsulation in the nanoparticles, which is much higher than the reports available¹⁷. Such high encapsulation efficiency of Lnd in LfNPs could be attributed to the strong hydrophobic interactions between Lf and Lnd. Taken together, the high drug encapsulation efficiency of LfNPs along with an optimum size and surface charge provides an increased advantage under in vivo conditions. The LfNPs may thus carry large amounts of drug to the site of action and increases the plasma half life of Lnd.

Figure 1C: Particle size and zeta potential of nanoparticles

Stability and biocompatibility of Lf nanoparticles:

Long shelf-life is pre-requisite to increase the translational potential of any formulation. Therefore, the storage stability of LfNPs was monitored by measuring their size over a period of 5 weeks. The nanoparticles showed no significant size change when stored under refrigerated conditions in PBS (pH 7.4), suggesting the high stability of LfNPs. The in vitro toxicity of the nanoparticles was assessed in B16F10 cells. Cells were treated with increasing amount of LfNPs (without Lnd) and cell death was analysed by PI uptake and subsequent flow cytometry analysis. The nanoparticles were found to be safe at each tested concentration.

pH dependent Lnd release studies:

To understand the release mechanism of Lnd from Lnd-LfNPs, a pH-dependent release study was carried out. Interestingly, it was found that approximately 60-80% of the entrapped Lnd was released at a pH of 5.0 to 6.0 which is similar to endosomal and lysosomal pH (Figure 2). The ability of LfNPs to release Lnd even at a mildly acidic pH as compared to neutral pH may facilitate a triggered release of Lnd in response to low pH encountered in the tumor environment and inside the endosomes. The low pH release of Lnd from Lnd-LfNPs may be attributed to structural changes in the Lf protein molecules constituting the nanoparticles. It has been reported by that lactoferrin undergoes a structural transition with an increase in α-helical content (by 30%) and decrease in β-sheet and random coil content (by 12%) as the pH is lowered from 7.4 to 5 ¹⁸. The pH triggered release of Lnd may provide an advantage in enhancing the pharmacological effects of Lnd by increasing its release specifically at the tumor site.

Figure 2: pH dependent release of Lnd

Enhanced antiproliferative activity of Lnd-LfNPs:

Lnd, the standard drug widely used for melanoma treatment is not effective because of the inability to achieve therapeutic doses at the target site due to its non-specificity. In addition, associated systemic toxicity greatly limits their therapeutic index which in due course leads to poor prognosis. A delivery system that can enhance the potency of drug by reducing its IC50 value would be desirable. The cytotoxic effect of Lnd-LfNPs was measured using MTT assay. The results suggest that Lnd-LfNPs exhibited more cytotoxic effect over all the experimental concentrations (Figure 3). Also, there was a significant decrease (3-fold) in IC50 value of Lnd-LfNPs (21.5 ± 2.56 μg ml-1) as compared to free Lnd (59 ± 3.0 μg ml-1). The enhanced cytotoxicity of Lnd delivered through LfNPs may be attributed to the higher intracellular concentration and prolonged retention of the drug achieved through delivery with LfNPs.

Figure 3: Antiproliferative studies of Lnd loaded nanoparticles

CONCLUSION:

The present work deals with development of a nano drug delivery system using lactoferrin protein for pharmaceutical use. The LfNPs developed in the study demonstrate a highly efficient delivery of Lnd specifically to melanoma cells. The Lnd-LfNPs is featured with high drug loading capacity, modest particle size (150 ± 20 nm) and narrow size distribution. A high Lnd release at mild acidic pH values, as observed in our studies is favorable for precisely targeting tumor cells and reducing toxicity to normal cells. Moreover, Lnd-LfNPs exhibited lactoferrin receptor specific cellular uptake, prolonged retention and enhanced cytotoxicity in melanoma cells. Thus our results suggest use of LfNPs as potential delivery vehicle to elevate the anticancer effects of chemotherapeutic drugs such as Lnd.

CONFLICT OF INTEREST:

None declared

ACKNOWLEDGEMENTS:

We express our sincere gratitude to the management of GITAM University, Visakhapatnam for supporting our work.

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Received on 20.08.2018 Modified on 10.09.2018

Research J. Pharm. and Tech 2018; 11(9): 4010-4014.

DOI: 10.5958/0974-360X.2018.00737.0

*Corresponding Author E-mail: mallinasrianusha@gmail.com