Lipid-polymer hybrid nanoparticles carrying doxorubicin and edelfosine combo for synergistic anticancer effect against drug-resistant Osteosarcoma

In this study, we have prepared a novel folate receptor-targeted doxorubicin (DOX) and edelfosine (ET)-loaded lipid polymer hybrid nanoparticle to enhance the anticancer efficacy in osteosarcoma. The dual-drug loaded nanoparticles showed a nanosized morphology and physiological stability. The targeted nanoparticles showed enhanced cellular internalization and subcellular distribution in MG63 cancer cells compared to that of non-targeted nanoparticles. Among many ratios of DOX and ET, 1:1 ratiometric combinations of drugs were observed to be highly synergistic in killing the cancer cells. MTT assay and caspase-3/7 activity assay clearly showed the superior anticancer efficacy of DE-FPLN formulations in inducing the cancer cell death. In vitro results indicate that the co-administration of two drugs in a folic acid-targeted nanoparticle could potentially induce the apoptosis and cell death. In vivo results displayed the potency of tumor cell killing and significant suppression of tumor growth without any detectable side effects. The lipid polymer hybrid nanocarriers with multiple properties of high drug loading, sequential and ratiometric drug release, improved physiological stability, prolonged blood circulation, and tumor-specific targeting are promising for the delivery of multiple drugs in the treatment of osteosarcoma.


Introduction
Osteosarcoma is one of the common forms of bone cancer accounting 60% of all bone cancer cases [1]. Osteosarcoma mainly affects children and adults of age been 10-18 years or within the first 2decades of their life. It mainly originates from mesenchymal cells and largely presents in large bones such as femur or tibia [2]. Later on, primary osteo cells spread to lungs making it difficult to treat. The recent development in technology and diagnosis did not improve the 5 year-survival rate which is still 65% making it a big cause of concern. Current treatment options include surgery followed by chemotherapy. Several high doses of anticancer drugs such as doxorubicin, cisplatin, ifosfamide were used to treat osteosarcoma, however, none led to satisfactory cure or treatment [3,4]. The failure of chemotherapy mainly arises from drug resistance and acute side effects of high dose of anticancer drugs [5]. Therefore, a novel strategy needs to be developed to improve the survival rate and therapeutic efficacy in osteosarcoma. 4 Combination of chemotherapeutic drugs is adapted as a standard therapy in the clinic compared to single drug administration. The combination of anticancer drugs acts in multiple pathways and effectively kills the cancer cells compared to that of single drugs which often result in failure or lead to drug resistance. In this regard, doxorubicin (DOX) is an anthracycline analog widely used for the treatment of osteosarcoma as well as multiple other cancers such as breast cancer or leukemia [6].
Despite being one of the effective drug available in the market, its anticancer efficiency often hampered by the development of multidrug-resistance (MDR) that led to the progression of recurrence of cancer tissues. The main reason behind MDR is believed to be ATP-binding cassette transporters such as p-gp that pumps the drug out of the cancer cells [7]. Moreover, anthracyclines are substrate for p-gp and therefore responsible for MDR and failure in the chemotherapy process and hinders its application at the clinical level [8]. In order to reverse these limitations, combination of a second agent was considered an appropriate strategy. Edelfosine (ET) has been reported to exhibit synergistic therapeutic effect in osteosarcoma and has been reported to significantly decrease the dose of DOX administration [9]. The alkyl-lysophospholipid (ET) has been shown to efficacious in many cancer cells. The mechanism of action involved with ET involves binding the lipid rafts of plasma membrane inducing extrinsic apoptosis pathways [10,11]. Also it induces the apoptosis via mitochondria and endoplasmic reticulum [12,13]. Two different therapeutic targets of nucleus and membrane are expected to improve the therapeutic efficacy and minimize the adverse effects [14].
However, when administered in the free form, several drawbacks such as poor bioavailability, rapid clearance from the systemic circulations are reported.
Lipid-polymer hybrid nanoparticles (LPN) have recently emerged as a potent alternative to lipid or polymer nanoparticles separately [15]. LPN is typically polymeric nanoparticles embedded by biocompatible lipid layers. Both drugs (DOX and ET) are stably loaded in the hydrophobic PLGA and lipid structures [16]. The protective lipid layers effectively decrease the release of encapsulated drugs in systemic circulation and thereby reduce the side effects to normal cells [17]. The presence of polyethylene glycol (PEG) on the lipid shell improves the stability of particles and prevents the absorption of serum protein and thereby prolongs its systemic circulation [18,19]. These properties 5 allow the accumulation of nanoparticles in the tumor tissues via a well-known enhanced permeability and retention (EPR) effect. In order to further increase the drug accumulation, targeting of nanoparticles to the tumor tissues would be beneficial [20]. In this regard, folate receptors (FR) which is overexpressed in the cancer cells binds specifically to the folic acid molecules [21]. The folate receptors are less expressed in the normal cells while overexpressed in the cancer cells and binds to folic acids in the nanoparticles [22].
The main aim of present study was to prepare DOX and ET-loaded lipid-polymer hybrid nanoparticles in order to increase the therapeutic efficacy in osteosarcoma. The dual-drugs were loaded in the LPN and characterized for wide range of in vitro and in vivo animal studies. The anticancer efficacy was tested in MG63 cancer cell-based xenograft tumor models. We have clearly showed that combination of DOX and ET significantly improved the anticancer efficacy.

Conclusions
In conclusion, we have prepared a novel folate receptor-targeted doxorubicin and edelfosine-loaded lipid polymer hybrid nanoparticle to enhance the anticancer efficacy in osteosarcoma. The dual-drug loaded nanoparticles showed a nanosized morphology and physiological stability. The targeted nanoparticles showed enhanced cellular internalization and subcellular distribution in MG63 cancer cells compared to that of non-targeted nanoparticles. Among many ratios of DOX and ET, 1:1 ratiometric combinations of drugs were observed to be highly synergistic in killing the cancer cells.
MTT assay and caspase-3/7 activity assay clearly showed the superior anticancer efficacy of DE-FPLN formulations in inducing the cancer cell death. In vitro results indicate that the co-administration of two drugs in a folic acid-targeted nanoparticle could potentially induce the apoptosis and cell death.
In vivo results displayed the potency of tumor cell killing and significant suppression of tumor growth without any detectable side effects. The lipid polymer hybrid nanocarriers with multiple properties of high drug loading, sequential and ratiometric drug release, improved physiological stability, prolonged blood circulation, and tumor-specific targeting are promising for the delivery of multiple drugs in the treatment of osteosarcoma.

Synthesis of DOX/ET-loaded lipid-polymer targeted nanoparticles
The dual-drug loaded lipid polymer nanoparticles were prepared by nanoprecipitation method. PLGA (10 mg), DOX (1 mg) and ET (1 mg) were dissolved in acetonitrile and mixed together. Then lecithin, DSPE-PEG (4:1 molar ratio) was dissolved in 5% ethanol aqueous solution at a weight ratio of 20% to that of PLGA mixture. The lipid fraction is heated at 65°C for 5 min. The PLGA/acetonitrile solution was gradually added in the lipid solution a dropwise manner to allow self-assembly process. The organic solvents were gradually removed at 37°C via a rotary evaporation method. The thus-formed nanoparticle was washed thrice with distilled water using a centrifugal device with a molecular weight cutoff of 3500 kDa. The nanoparticle was suspended in distilled water and stored in 4-8°C until further use. As much as possible, drug-loaded nanoparticles were prepared fresh for all the experiments. The drug-loading capacity and drug-loading efficiency were calculated using the same centrifugal tubes.
The filtrate containing the free drug was evaluated by HPLC method and plotted.

In vitro drug release study
The drug release of DOX and ET from DE-LN and DE-FLN was studied using a dialysis tube (Spectra/Pore, MWCO 3500). The study was performed at 37°C in a regular shaker (100 rpm). The dual drug-loaded nanoparticle dispersions were sealed in a dialysis tube and placed in a 30 ml of acetate buffered saline (ABS, pH 5.0) and phosphate buffered saline (PBS, pH 7.4) in a Falcon tube.
The tube was placed in a shaker bath at a speed of 100 rpm/min and maintained at 37°C. At predetermined time, small aliquots of samples were collected and replaced with equal volume of the fresh sample to maintain the sink conditions. The amount of drug released in the buffer was calculated using HPLC method. The HPLC system (Waters, Milford, MA, USA) consists of Waters 1525 Binary HPLC pump, a Waters 2707 Autosampler, and a Waters 2475 multi λ fluorescence detector. A column consists of C-18 column (150 mm × 4.6 mm, 5 µm particle size, and 100 Å pore size, AkzoNobel/Kromasil, Brewster, NY, USA) was used at a room temperature. Flow rate was maintained at 1 ml per minute.

In vitro cell culture
MG63 osteosarcoma cells were cultured in RPMI-1640 growth medium supplemented with 10% FBS 7 and 1% antibiotic mixture. The cells were grown in ambient conditions of 5% carbon dioxide and moisture status.

In vitro cellular uptake
The targeting ability of nanoparticles towards MG63 cells were studied by confocal laser scanning microscopy (CLSM). The 6-well plate was covered with cover slip and cells were seeded in the capacity of 2×10 5 cells per well and incubated for 12h. Next day, cells were exposed with LPN-loaded with fluorescent Nile Red at a concentration of 0.25 mg/ml and incubated for 2h. The cells were washed and fixed with 4% paraformaldehyde and incubated for 10 min followed by staining with DAPI solution for 5 min. The cells were well-rinsed with PBS and observed under confocal microscope (Zeiss LSM 710; Carl Zeiss Meditee AG).

In vitro cytotoxicity assay
The in vitro cytotoxicity assay in MG63 cancer cell was evaluated by MTS as per the guidelines of manufacturers of CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay (Promega, Madrid, Spain). In brief, 8000 cells were seeded in the each well of 96-well plate and incubated for 24h. After that, cells were treated with different concentrations of free drugs and free dru combination and drugloaded nanoparticles and incubated for 24h. After 24h, supernatant and all media was removed and washed twice with PBS. The cells were then added with 100 µl of complete media containing 15µl of MTS solution and incubated for 4h. The absorbance was measured using a microplate reader (iEMS reader MF, Labsystem, Helsinki, Finland) at a wavelength of 690 nm. The IC50 value calculations and statistical analysis was performed using GraphPad Prism software. The combination index was calculated using CalcuSyn software (Biosoft, Cambridge, UK). A CI value of less than 0.9 indicates synergism while 0.9-1.1 indicates additive and anything above 1.1 indicates the antagonistic characteristics.

Caspase 3/7 activity
The caspase-3/7 activity was evaluated using a Caspase-Glo-3/7 assay kit (Promega, USA) as per the manufacturer's protocol. In brief, 8000 cells were seeded in the each well of 96-well plate and 8 incubated for 24h. After that, cells were treated with different concentrations of free drugs and free drug combination and drug-loaded nanoparticles and incubated for 24h. After 24h, supernatant and all media was removed and washed twice with PBS. The caspase activity was measured by adding the assay reagent in a proportion of 1:1 to each well. The luminescence signal was measured using an Tecan GENios microplate reader (Tecan Group Ltd, Switzerland) after incubating for 30 min post reagent addition. The experiments were performed in triplicate. 150 µL of plasma was mixed with 150 µL of acetonitrile for 30 min. The samples were then centrifuged at 12,000 rpm for 10 min and 10 µL of the supernatant was injected into the HPLC system for DOX and EDL analysis.

In vivo antitumor efficacy
The BALB/c female mice with an average weight ranging from 20-22 gram were purchased and given humanely care with free access to food and water. All the animal studies are performed in compliance with the Institutional Animal Ethical Care and Management (IAECM) guidelines, The Eighth People's Hospital of Shanghai, China. In brief, MG63 cells were grown in the culture medium and after 80% 9 confluence; cells were harvested from the culture flask and subcutaneously implanted in the right flank of the mice in the capacity of 5×10 6 cells/mouse. The tumor growth was closely observed and the experiment was initiated when the tumor volume reaches approximately 100 mm 3 . The mice were divided into 6 groups with 8 mice in each group. The mice groups were administrated with free DOX, ET, DOX/ET cocktail, DE-PLN and DE-FPLN at a maximum dose of 5 mg/kg of mice body eight for 3 times. All mice were tagged and tumor volumes were measured at predetermined time interval using the tumor volume (V) calculation formula V=a×b×1/2 whereas 'a' and 'b' are respective long and short diameters of the tumor. The tumor size was measured using an Vernier caliper.

Histological analysis of tumors
The tumors were surgically removed from the mice after sacrificing it humanely following all the animal handling protocols. The tumors were immediately fixed in 10% formalin for 2h and embedded in a paraffin in a thickness of 5 mm. The tissue section is then subjected to H&E staining and observed through the fluorescence microscope (Olympus, NIKON).

Statistical analysis
All data are performed as the mean ± standard deviation. All statistical analysis was performed using SPSS 10.0 software (SPSS Inc., Chicago, USA). A statistical difference of p<0.05 was considered significant.

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  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 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 drugloaded nanoparticles (targeted and non-targeted) was evaluated in MG63 osteosarcoma cells. As seen

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 coadministration 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].  [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,

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 16 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.     In vivo toxicity analysis in animal model. Acute toxicity analysis of free drugs and drugloaded formulations in vital organs included heart, liver, spleen, lungs and kidney by H&E staining analysis. The toxicity of DOX in heart is highlighted by arrow marks which showed disrupted cardiac muscle fibres and cytoplasmic vacuolization. Liver and kidney of DOXtreated group showed signs of marked degenerative changes.

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