Formulation and optimization of DOX/ET-loaded folate-targeted lipid polymer nanoparticles
The schematic presentation of preparation of DOX/ET-loaded lipid polymer hybrid nanoparticle is presented in Figure 1. The DOX and ET along with PLGA were dissolved in organic solvent and stirred. The lipid mixture was added to the polymeric solution and allowed to form the self-assembly resulting in the creation of lipid-polymer hybrid nanoparticles. The hydrophobic nature of the drugs allowed the stable incorporation in the lipid matrix and hydrophobic PLGA. Earlier, studies have shown that the presence of PEG on the outer surface will ensure the protection from reticuloendothelial (RES) based clearance system that could potentially result in the prolonged blood circulation and reduced non-specific binding. The presence of folic acid moiety will confer the targeting capacity to the nanoparticles towards the tumors tissues in the body.
The particle size and surface charge characteristics were evaluated by dynamic light scattering (DLS) analysis. The mean particle size of DE-FPLN was observed to be 122.5±2.45 nm with a narrow distribution index of 0.140 (Figure 2a). The average surface charge of DE-FPLN was observed to be -18.4±1.26 mV. The particle size was further confirmed by the TEM imaging (Figure 2b). All the particles perfectly spherical in shape and uniformly spread over the copper grid. Especially, a greyish shell was observed at the periphery of each particle that might be due to the presence of lipid shell. A particle in the nanosize ranges deemed suitable for the tumor targeting as it can penetrate the tumor tissues via enhanced permeation and retention (EPR) property. It is reported and wide recognized that tumors possess leaky vasculature coupled with poor drainage access which allows the high accumulation of particles and intracellular concentrations when the particles are nanosized [22]. The loading capacity of DOX in DE-FPLN was 6.89% w/w while ET was 5.75% w/w, respectively.
In vitro drug release
The release of DOX and ET from DE-FPLN under different pH conditions was evaluated by dialysis method. As seen (Figure 2c), differential release pattern was observed under physiological and acidic pH conditions. The release rate of drugs was higher in acidic conditions compared to that of in pH 7.4 conditions. For example, ~18% of DOX released in 24h in pH 7.4 conditions compared to ~32% in pH 5.0 conditions. Similarly, 12% of ET released in 24h in pH 7.4 conditions compared to nearly 25% ET release in pH 5.0 conditions. It should also be noted that release rate of DOX was relatively higher than that of the release rate of ET in both pH conditions. A differential release pattern for both the drugs might be attributed to the encapsulation of one drug in polymer core and another in lipid matrix or vice versa. Importantly, both the drugs showed a sustained release of drug from the nanoparticles. The release trend continued until the end of the study period with higher release in acidic media mimicking the tumor microenvironment. This type of sequential release of drugs in the tumor cells results in effective synergistic therapeutic effects. Besides, enhanced drug release in lower pH conditions compared to that in physiological conditions boosts the prospect of higher drug release in the lysosomal/endosomal pH conditions in the tumors [23].
Intracellular uptake in MG63 osteosarcoma cells
The targeting ability of the nanoparticles (DE-PLN and DE-FPLN) in MG63 cancer cell was evaluated by confocal laser scanning microscopy (Figure 3). CLSM images showed a red fluorescence from Nile red-loaded nanoparticles in the lysosomal regions of cancer cells. Data clearly reveal that folic acid-targeted nanoparticle (DE-FPLN) showed a remarkably stronger red fluorescence compared to that of non-targeted DE-PLN indicating that the receptor-mediated internalization via FR-mediated endocytosis might be responsible for enhanced accumulation of particles. FR targeting played a seemingly crucial role in enhancing the cellular uptake of nanoparticles by MG63 cells. Accumulation of non-targeted nanoparticles in the cancer cells further highlight the fact that other mechanisms such as clathrin or caveolae-mediated could be associated with the nanoparticles uptake. It is believed that nanoparticles enter the acidic lysosomes and the nanoparticles get destabilized and results in the release of encapsulated drugs. These drugs then reach their targeted location inside the tumors. The cellular internalization of nanoparticles was further confirmed after pretreatment with free folate. As shown, marked reduction in cellular uptake of DE-FPPLN was observed in cells pretreated with free folate indicating the expression of folate receptors and receptor-mediated cell uptake in the cancer cells (Figure S1).
In vitro cell viability – Combination Index
Administration of single drugs often resulted in poor efficacy and multidrug resistance (MDR). In this regard, combination chemotherapy has been proven to be remarkably effective than single drugs. The use of multiple drugs will act on the different pharmacological pathways and prevent the drug resistance and improve the therapeutic efficacy. It is well known that specific ratio of drugs is particularly more effective while certain ratios are ineffective. In order to evaluate the perfect ratio of two drugs, drugs were combined in different ratiometric combination and cell viability was evaluated. As seen (Figure 4a), different ratiometric combinations have different effect on the cell viability of cancer cells. The therapeutic effect of combinations of drugs are evaluated in terms of synergistic, additive and antagonistic. The Chou and Talalay method-based CI tells that the values below <0.9 indicates the drug synergy while CI between 0.9-1.1 indicates additive nature and >1.1 indicates the antagonistic effect of the drugs. As seen, DOX:ET were highly synergistic in 1:1 w/w ratio indicating a maximal synergistic interaction between the drugs. The DOX:ET at 1:2 and 2:1 though synergistic, it was less compared to that of 1:1 ratiometric combinations. On the contrary, drug ratios of 5:1 and 10:1 were antagonistic in nature. Based on the C1 value, we have chosen DOX:ET to study further for all the in vitro and in vivo studies.
In vitro cytotoxicity assay
Following the ratiometric analysis of DOX and ET, in vitro cytotoxic effect of free drugs and drug-loaded nanoparticles (targeted and non-targeted) was evaluated in MG63 osteosarcoma cells. As seen (Figure 4b), all the formulations showed a typical dose-dependent cytotoxic effect in the cancer cells. The cell viability of cocktail DOX:ET (DE) treated cells were significantly lesser compared to that of single drugs either DOX or ET alone further suggesting potential of combination of drugs. It should be noted that dual-drug loaded nanoparticle showed relatively higher anticancer effect than the free combination of drugs. Importantly, folate receptor-targeted DE-FPLN showed a remarkable anticancer effect in the MG63 cancer cells. The cytotoxicity profiles were fitted in logistic model to calculate the IC50 value - a concentration required to kill 50% of the cancer cells. The IC50 values of DOX, ET, DE, DE-PLN and DE-FPLN stood at 4.5 µg/ml, 6.4 µg/ml, 2.3 µg/ml, 1.08 µg/ml, and 0.26 µg/ml, respectively. Data clearly reveal the superior anticancer potential of DE-FPLN formulation in controlling the proliferations of cancer cells. DE-PLN though showed notable decrease in cell viability due the combination therapeutics, DE-FPLN showed the significantly lower cell viability attributed to the higher cellular internalization of targeting ligand-based carrier system.
Caspase-3/7 based apoptosis activity
The mechanism of cell death was evaluated in terms of cell apoptosis using caspase-3/7 activity assay in MG63 cells. For this purpose, MG63 cells were exposed with different formulations and incubated for 24h. As seen (Figure 5a), caspase-3/7 activity of cocktail DE remarkably increased compared to that of single drug treatment. Consistent with the cell viability assay, DE-PLN showed higher caspase-3/7 activity than that of free drug cocktails. Interestingly, DE-FPLN showed 1.5-fold and 3-fold higher caspase-3/7 activity compared to that of DE-PLN and DE cocktail, indicating the co-administration of two drugs in a folic acid-targeted nanoparticle could potentially induce the apoptosis and cell death. Such enhancing the cytotoxic activity between DOX and ET has been reported in Ewing’s sarcoma cells that could be attributable due to different mechanism of actions [24]. DOX is reported to exert its pharmacological effect by the inhibition of DNA synthesis in nucleus while ET targets the cell membrane, mitochondria and endoplasmic reticulum [25,26].
Biodistribution and Pharmacokinetic analysis
A biodistribution study of Cy5.5-based DE-PLN and DE-FPLN was performed in xenograft animal model following the i.v. administration. As shown by whole body image and ex vivo organ image, DE-FPLN exhibited preferential accumulation in tumors compared to that of DE-PLN indicating the tumor targeting ability of DE-FPLN (Figure S2). A notable accumulation of DE-PLN in the tumor might be attributed to the passive accumulation by EPR effect, however, relatively lower fluorescence intensity was observed compared to that of DE-FPLN. It must be emphasized that vital organs including heart, kidney and lungs pretty much showed similar levels of nanoparticle uptake.
The plasma concentration-time profiles of DOX and EDL following single dose intravenous (IV) administration of free form and nanoparticle encapsulated form are presented in Figure 5b. Free DOX, EDL and DE-FPLN (DOX/EDL) were administrated at a dose of 10 mg/ kg via the femoral vein. Free DOX and EDL were rapidly cleared from the circulation within 4 h of IV administration and exhibited linear pharmacokinetics. As reported previously, the linear pharmacokinetic profile of DOX remained the same whether administered at high or low dose. In contrast, DOX and EDL had a remarkably prolonged plasma circulation time after administration of DE-FPLN (DOX/EDL) and maintained a therapeutic drug level throughout the study period. For example, 0.121±0.25 µg/ml of free DOX was observed after 4h of i.v administration whereas 0.256±0.21 µg/ml of DOX was observed from nanoparticle formulation after 24h indicating the superior potential of carrier-mediated delivery system. The prolonged blood circulation profile of DE-FPLN was mainly attributed to the shielding effect of PEG, the excellent stability of the carrier/drug formulation in blood circulation, and the negative surface charge of the nanoparticles. Additionally, nanosized particles might be able to evade the macrophage system in the systemic blood circulation. The presence of a PEG outer shell in the micelles reduced the effective surface charge and increased the hydrophilicity that would decrease the liver and splenic uptake.
In vivo antitumor efficacy
The therapeutic efficacy of formulations was evaluated by the intravenous administration for formulations in tumor-bearing mice model. The mice were administered with DOX, ET, DE, DE-PLN and DE-FPLN, respectively. As shown (Figure 6a), free DOX and ET did not have much effect on the tumor growth while cocktail combination DE showed obvious tumor regression compared to single treatment and control. Importantly, DE-FPLN outperformed all the other groups and significantly delayed the tumor progression in the animal models. DE-FPLN showed 1.5-fold lower tumor volume than DE-PLN while it showed 2-fold lower tumor volume and 3-fold lower tumor volume for DE and single drugs, respectively. The main reason behind the excellent antitumor efficacy might be due to prolonged blood circulation and active targeting of the nanoparticles to the tumor tissues. The folate receptors overexpressed in the tumors will bind specifically to the folic acid ligand conjugated on the nanoparticle surface and results in enhanced accumulation in the local area and high therapeutic efficacy. Significant differences in tumor volume were observed between mice treated with 2.5 mg/kg and 5 mg/kg, however, insignificant difference was observed at higher doses (Figure 6b). Except at the last time point, mice treated with 5 mg/kg and 10 mg/kg was insignificant. The experiment further reiterates the fact that 5 mg/kg of the combination of DOX and EDL could be appropriate. Besides, hallmarks of Tumors are characterized by the presence of poor lymphatic drainage and leaky vasculature that allows the internalization of nanocarriers sized up to ∼200 nm specifically into the tumoral regions, commonly referred to as EPR effect. The features of improved physiological stability and prolonged blood circulation contributed to selective accumulation and long-term retention of DE-FPLN at the tumor sites and enhanced therapeutic efficacy. Fernandez et al, have reported a synergistic effect of DOX and EDL. The authors reported that the synergistic effect was well-correlated with Caspase-3/7 activity indicating a caspase-mediated cell death [27]. Other authors also observed the combination effect of DOX and EDL in Ewing's sarcoma cells [28]. DOX is known to exert its effect by inhibition of DNA synthesis in nucleus, while, EDL targets the membrane of the cells, endoplasmic reticulum and mitochondria. Stability and suitability of carriers also played an important role in enhancing the anticancer effect of encapsulated drugs. For example, Zhang et al reported the enhanced anticancer effect employing similar polymer-lipid hybrid nanoparticles [29]. Authors have reported that polymer-lipid hybrid nanoparticles improved the pharmacokinetic profile of dual drugs (DOX and Mitomycin C) and enhanced the antitumor effect in xenograft breast cancers.
H&E analysis revealed that DE-FPLN exerted remarkable damage to the tumor tissues compared to any other administered groups (Figure 6c). DE-FPLN treated tumors exhibited pyknotic cells in which nuclei were condensed which are seemingly signs of apoptosis and dead cells. Furthermore, mice treated with DE-FPLN did not show any significant changes in the body weight throughout the study period indicating a lack of any toxic effects. In contrast, mice treated with free DOX and DE resulted in 10% shedding of body weight which is consistent with the other reports of toxicity (Figure 6d). In case of DE-FPLN, low dosage of both drugs which is stably incorporated in the nanoparticles was beneficial in reducing the side effects and highlights the superior advantages of nanocarrier system. The significant improvement in the antitumor efficacy stem from the fact that DE-FPLN delivers the drug to the tumor tissues in a specific ratiometric manner that allows the synergistic killing of the cancer cells and tumor regression. In contrast, free drug and cocktail combinations (DE) may not equally penetrate the tumors and results in subpar therapeutic effect.
Acute toxicity analysis
Drug-related toxicity to major organs was evaluated by H&E staining. Free DOX treatment to the animals resulted in serious damage to the liver, kidney and heart (Figure 7). EDL was relatively less toxic compared to that of DOX as observed from the H&E image. Hepatic lesions were observed with severe atrophy of hepatic cells. The free DOX resulted in acute cellular swellings of the liver and the mean hepatocyte diameter for the free DOX-treated group was 26.18 ± 2.16 µm compared to 14.25 ± 1.68 µm in the non-treated animal group. Cardiac H&E revealed that free DOX resulted in focal rarefactions, focal areas of disrupted cardiac muscle fibers and cytoplasmic vacuolization. In contrast, DE-FPLN showed no myocardial degeneration and was devoid of any cardiotoxicity. The kidney in DE and DOX-treated group showed notable congestion with marked degenerative changes, and disrupted epithelium. The sections of liver in DE and DOX-treated group showed marked central vein congestion, marked bile duct hyperplasia, and dilation of sinusoidal spaces, and some sections showed marked degenerative changes. The H&E stained sections of liver and kidney from formulation-treated group appeared normal suggesting lack of any hepatotoxicity or nephrotoxicity. No such damage or sign of toxicity was observed in DE-FPLN treated animal group suggesting the efficacy of carrier-based drug delivery. The ability of the carrier system to decrease the organ damage and increase the anticancer efficacy is of great significance.
Despite the excellent finding in this investigation, study is not without the limitations. It was assumed in this study that a higher dose would confer greatest efficacy, however, in terms of toxicity, maximum tolerable dose (MTD) is not determined. The acute toxicity effects monitoring performed in the current study was done only at the dose range tested, but not at the MTD of the various compounds involved (as the MTDs have not been determined). Future studies will focus on quantifying dose-response effects, with simultaneous emphasis on MTD. Furthermore, we would like to continue the study with different targeting ligand or combination of two or more ligands.