pH Sensitive Lipid-Polymer Hybrid Nanoparticles Mediated Delivery of Docetaxel: A Viable Approach for Breast Cancer Therapeutic Intervention Development

Present study was planned for the development of pH sensitive lipid polymer hybrid nanoparticles (pHS-LPHNPs) loaded with docetaxel (DTX) for guided and target specific cytosolic-delivery delivery of docetaxel (DTX). pHS-LPHNPs were formulated to entrap DTX by self-assembled nano-precipitation technique and characterised with respect to zeta potential, particle-size, entrapment efficiency, PDI as well as invitro drug release. The cell viability, apoptosis, cellular-uptake, pharmacokinetics, biodistribution in vital organs, % changes in tumour volume and survival of breast cancer bearing animals were used for the evaluation of efficacy of the formulation. In-vitro studies showed increased cytotoxicity at lower IC 50 and better cellular-uptake of pHS-LPHNPs mediated drug by breast cancer cell lines. We saw the better rate of apoptosis of breast cancer cells via Annexin V/Propidium iodide staining. Moreover, in-vivo studies demonstrated improved pharmacokinetics and targetability with minimum drug circulation in deep-seated organs upon delivery of DTX via pHS-LPHNPs in comparison with LPHNPs-DTX and free DTX. We observed sizeable % reduction in tumour-burden with pHS-LPHNPs-DTXthan that withLPHNPs-DTX &free DTX. In brief, pHS-LPHNPs mediated delivery of DTX exhibited promising approach for developing therapeutic-interventions against breast-cancer.


Introduction
The novel drug delivery systems (NDDS)holds importance in developing therapeutic interventions against cancers,cardiac, and infectiousdiseases [1,2].The novelnanoparticles show advantages over other conventional therapeutic approachesby means of structural rigidity, targeted &controlledrelease and longer shelf-life [3]. However, systemic administration of polymeric nanoparticles is not recommended due to their swift uptake by the reticuloendothelial system (RES) [4][5][6].This rapid uptake of polymeric nanoparticles by REShas been refrained by conjugatingthe lipids and polymerswith polyethylene glycol (PEG) [7][8][9][10]. PEG was used to conjugate to a bio-specific ligand that could target and help deliver the entrapped content from the nanoparticles to the preferred site of action [11][12][13][14][15].The effective delivery at the preferred site and cytosolic delivery of drugsare important for effective treatment [16,17].The intracellular uptake might be facilitated via some internal ligands i.e. Folate [16], transferrin [18], lectins [19], Peptides like TAT [17]. The ligand anchored nanoparticles mediated delivery should to be able to regulatebiological functions required under the influence of local stimuli characteristicssuch as increased temperature or low pH of targeted pathological sites [20].Therefore, delivery system with an extended circulation timeto achieving significant enhanced permeability and retention(EPR) effect. This directed to the accretion of drug loaded nanoparticles at the target sites, and fusogenic properties facilitate fusion of drug delivery system (DDS) with cell membranes, and allowuptake by the target cells and destabilization of endosomes [21].The controlled cytosolic delivery of bioactives is one of the majorhurdles in the drug delivery for developing treatment alternatives against tumours. Therefore our work reports the development of pH sensitive lipid polymer hybrid nanoparticles (pHS-LPHNPs) to deliver drugs into the cytosol directly to increase the drug efficiency by avoiding the endocytic pathwayand rendersprotection of drug from the lysosomal degradation.
The issues associated with the delivery of DTX may be overcome by the encapsulation of DTX into pHS-LPHNPs to achieving the selective cytotoxicity in the breast cancer therapy. Thus we decided to explore the comparative delivery potential of DTX encapsulated pH sensitive LPHNPs with non-pH sensitive LPHNPs for developing effective treatment strategies against breast cancer [12].
Our group developed a one step, self-assembled pHS-LPHNPs to ameliorate the encapsulation and cytosolic delivery of hydrophobic drug (DTX) for the effective cancer therapy [10]. In brief, present study shows the evaluation of anti-tumour properties of DTX-loaded pHS-LPHNPs in breast cancer cells i.e. MDA-MB-231 & MCF-7. Further, in vitro findings were complemented well by the data obtained in an experimentally inducedbreast cancer animal model. We believe our resultssurface the targeted and controlled drug delivery potential of pHS-LPHNPs to improve the therapeutic effectiveness of DTX. were procured. Lutrol® F-87 was obtained as sample from BASF, Mumbai, India. All other chemicals and reagents were of analytical grade unless otherwise specified.

Formulation of pHS-LPHNPs
DTX encapsulated pHS-LPHNPs (pHS-LPHNPs-DTX)were prepared following the method reported by our group [10]. In brief, DOPE and oleic acid (4:1) was dissolved in DMF and heated to 65-70 °C with constant stirring at 500 rpm (Remi, Mumbai, India). The PLGA polymer and DTX were dissolved in DMF at 1 mg/mL. Both lipid and polymer solution were mixed together then mixed with the surfactant solution (0.5%) at the speed of 1 mL/min with moderate stirring and 1 mL of water was added drop wise to the solution. It was stirred for 2 h for the evaporation of the organic solvent. The pHS-LPHNPs were collected, washed and re-suspended in water. To prepare simple LPHNPs, DOPE&Oleic acid was replaced by Egg PC and Cholesterol using the same method as above.
The height and diameter of the pHS-LPHNPs was done by atomic force microscopy (AFM, Multimode-8 HR, Bruker USA). In this method, pHS-LPHNPs (10 µl) were carefully put on a silicon wafer. De-ionized water was used to remove the excess stuff from the surface. Then the sample was dried to create a thin film and scanned by placing under the lens of AFM, and three dimensional structures were observed [31].
Percent drug entrapment (%DE) was calculated by using lysis technique. In brief, lyophilized pHS-LPHNPs in predetermined quantity of were mixed with 5 mL solution of ACN: H 2 O (6:4) and sonicated for 3 min. The solution was filtered and further diluted with ACN: H 2 O(6:4) and charged onto HPLC system (e2695, Waters, Milford, USA) [32]. Percent drug entrapment (%DE)and percent drug loading (%DL) were estimated by using thefollowing equations [11] 2.4 In vitro DTX release from pHS-LPHNPs To evaluate the pH sensitivity of prepared pHS-LPHNPs, the in vitro DTX release in two different buffered saline of pH 7.4 & 5.5 was investigated bythe dialysis tubing. In Brief, 2 mL of pHS-LPHNPs-DTX formulation was kept in a dialysis bag (MWCO 10 kDa) that was stirred at 100 rpm in the dialysis solution at 37 ± 1 °C [10]. At pre-determined time schedule, 0.5 mL of medium was taken out and same quantity of fresh medium was added at each withdrawal. These samples were assayed to estimate the drug contentby HPLC at 230 nm [33].

pH induced aggregation of pHS-LPHNPs
Aggregation of pHS-LPHNPs due to low pH was observed by the estimation of particle size. About 50 µL of pHS-LPHNPs was mixed to 5 mL of 7.4 and 5.5 pH bufferseach and incubated at 37 ± 1 °C. Samples were taken and particle size was measured atpredetermined time intervals.

2.6
In vitro Studies 6 2.6.1 Cell culture Human breast cancer cell lines i.e. MDA-MB-231 &MCF-7 cells were cultured and maintained with minor modifications in the protocols reported elsewhere [10,34]. In brief, both the cell lines were cultured in 5% CO 2 atmosphere at 37 °C in Dulbecco's modified eagle media (DMEM). The cells were harvested with 0.25% trypsin and 1% EDTA solution upon reaching 80% confluency and diluted as per the requirement.

Cell viability studies
About 1 × 10 4 cells/well in 96 wells culture plates were seeded (Tarsons Products Pvt Ltd, Kolkata, India) [12,34]. After 24 hr, culture media was replaced with the medium containing the DTX, LPHNPs-DTX, pHS-LPHNPs-DTX to the respective wells at concentration equivalent to 0.05, 0.1, 1, 10, and 20 µg/mL of free DTX and incubated for an additional 24, 48 and 72 hr. The wells were flushed with physiological PBS and then 100 µL of MTT solution (1.25 mg/mL in PBS) was added to each well followed by re-incubation for 4 hr to allow the formazan crystals formation. About 200 µl of DMSO was added to each well to solubilize the formazan crystals. The untreated cells were taken as control. The absorbance (Abs) of obtained solution was recorded at 570 nm by 96 well plate reader (BioTek instruments, USA). The cell viability was calculated by using the following formula [35]. CalcuSyn software was used to calculate IC 50 values via median-effect plot.

Qualitative and quantitative cell uptake studies
The in-vitrostudies for cell uptake wereperformedas described elsewhere [12]. The qualitative cell uptake ofpHS-LPHNPswithMDA-MB-231&MCF-7cellswasestimatedby using confocal microscopy (CLSM).The cells were inoculated on coverslip (Tarsons Products Pvt Ltd, Kolkata, India) about 5 × 10 4 cells/well placed in 6-well plate and incubated for 24 hr at 37 °C. Cells were incubated with pHS-LPHNPs (1 µg/mL equivalent to C6) for 2hr at 37 ± 1 °C followed by washing with PBS (three times) to eliminate the extracellular particles. Cells were fixed with 4% PFA for 20 min at 20 °C.The coverslips were fixed on slides and observed with CLSM (eclipse Ti, Nikon).
The quantitative evaluation of cell uptake was conducted by HPLC analysis of DTX [33]. Both the cells were inoculated (5 × 10 4 cells/well) in 24 wells plates. These were incubated overnight to allow the cells to adhere. Growth media was replaced with media containing pHS-LPHNPs-DTX (equivalent to 10, 20, 30, 40 µg of DTX) and again incubated for 2hr to see the effect of concentration on cellular uptake. The washing of cells (3 times) were carried out with PBS and lysed with 0.1% Triton™ X-100 after incubation. The internalized DTX was extractedwith ACN: H 2 O (6:4) and cell lysate was centrifuged for 15 min at 25,000 rpm. The supernatant was analysed by HPLC.

In-vivo study
All the experimental protocols were approved by Institutional Animal Ethical Committee of Jilin University, Changchun, China.The animal care and experimental protocol were followed as per the guidelines of the animal ethics committee. The pharmacokinetic, bio-distribution, and antitumorstudies were performed as reported earlier [12,37]. 25-30gBALB/c female micewere acclimatized at 25 ± 1 °C and 45 − 55% RHwith natural day/night conditions with ad libitum food and water for 7 days before the commencement of experiments. In order to develop a xenograft model, approximately 4 × 10 5 MDA-MB-231 cells with 50% matrigel in 50 µL of culture medium injected subcutaneously into the mammary fat pad of female mice [38].
Tumor volume was measured by the following equation.
Where length and width of the tumour was measured by a Vernier calliper 2.7.1 Pharmacokinetics study Animals were kept in 3 groups consisting 6 animals each. The group I, II and IIIwere injected asingle dose of free DTX, LPHNP-DTX &pHS-LPHNP-DTX (equivalent to 10 mg/kg of DTX) through intravenous route.200µlof blood wasdrawn through the puncture of retro-orbital sinus at regular time intervalswith the partial anaesthesia and stored at-20 °C until analysed. 300 µl acetonitrile (ACN)was added to 100 µl of plasma to remove proteins, then centrifuged to obtainsupernatants and analysed for DTX [33]. Thekinetica software (Thermo-scientific, USA) was used to study pharmacokinetic parameters such as peak plasma concentration (C max ), half-life (t 1/2 ), area under the curve (AUC), mean residence time(MRT), and time to achieve maximum plasma concentration(T max ).

Bio-distribution study
Bio-distribution of drug was estimated on xenograft BALB/c female mouse model. Animals were divided in 3 groups with 6 animals each. After 24 hr of single dose administration(DTX, LPHNPs-DTX and pHS-LPHNPs-DTX),animals were euthanized and highly perfused organs like liver, heart,kidney, spleen, lungs and tumor were removed and weighed. 20% tissue homogenate was prepared in normal saline and stored at -20 °C. 300 µL of ACN was added to 100 µL of tissue homogenate to remove the proteins followed by centrifugation, filtrationand analysis [33], and the distribution pattern was evaluatedin highly perfused organs.

In Vivo anti-tumour activity
The anti-tumor activity of pHS-LPHNPs-DTXwereevaluated in experimentally induced breast cancer mouse model [12].Animals were randomly distributed into 4 groupsconsisting 6 animals each. Group 1, 2, &3 received DTX, LPHNPs-DTX andpHSL-PHNPs-DTX, respectively. The 4th group received normal saline and served as an experimental control. Formulations (equivalent to 6 mg/kg DTX) were administeredthrough lateral tail vein twice a weekfor 5 weeks,and tumor size wasmeasured after 5 weeks of treatment.
3 Results And Discussion 3.1 Preparation and characterization of pHS-LPHNPs formulations DTX encapsulated pHS-LPHNPs were prepared using one step, self-assembled nano precipitation techniques [41] (Schematic).
The DTX were encapsulated in the developed pHS-LPHNPs in PLGA polymeric core [10,24]. This core was encircled by crust of semi-polar biocompatible phospholipids along with DOPE and DSPE-PEG layer [41].
The average particle size of LPHNPs-DTX and pHS-LPHNPs-DTX was estimated 126.36 ± 4.67 nm and 151.31 ± 6.19, respectively. PI (< 0.18) submits the uniform distribution of pHS-LPHNPs-DTX, and zeta potential of pHS-LPHNPs-DTX formulation was calculated to be -12.31 ± 0.95 (Table 1). Table 1 Formulations were characterized with respect to particle size, PDI, zeta potential, entrapment efficiency, and drug loading The atomic force microscopy (AFM) and field emission scanning electron microscopy (FE-SEM) observations of these formulations confirm the spherical shape and nanometric size of formulated nanocarriers (Fig. 1).However, minor difference was seen in particle size when analysed by zetasizer.

In-vitro drug release
In-vitro drug release of pHS-LPHNPs-DTX& LPHNPs-DTX was carried out with two different pH buffers (pH 7.4 &5.5). As expected, DTX % release from pHS-LPHNPs was seen greater (42%) at low pH (5.5) within 12 hr whereas only 20% atpH (7.4). This release was trailed by slow and sustained for next 108 hrs.The drug release pattern from LPHNPs-DTX was nearly same but showed minordifference in the cumulative percent of drug release at both pHs. Fast DTX release from pHS-LPHNPs in acetate buffer (pH 5.5) was observed that may be due to the rapid destabilisation of lipid adsorbed atthe outer surface of the PLGA core of pHS-LPHNPs and release of entrapped drug from the polymeric core. The release of DTX from pHS-LPHNP-DTX was significantly (p < 0.05) higher as compared to LPHNPs-DTX upto 120th hr (Fig. 2).These findings are in accordance to the previous studies [10] and validate our findings.

pH Induced pHS-LPHNPs aggregation
The aggregation of the prepared pHS-LPHNPs-DTX was assessed by the changes seen in particle size and charge due to changes observed in pH.The particle size of pHS-LPHNPs-DTX was seen increased considerably at low pH DTX&LPHNPs-DTX (Fig. 4).

Qualitativeand Quantitative assessmentof cell uptake
The comparative analysis of intra-cellular uptake of coumarin-6 encapsulated LPHNPs (LPHNP-C6) and pH sensitive LPHNPs (pHS-LPHNPs-C6) was done. The correlation of cell uptake with cytotoxic effect was calculated in MCF-7 & MDA-MB-231cells. LPHNPs-C6 and pHS-LPHNPs-C6 incubated with both the cells for 2hr for qualitatively assess the rapid internalisation (Fig. 5). The fluorescent signals emitted by LPHNPs-C6 & pHS-LPHNPs-C6 formulations were quantified more than that seen with free C6 formulation (Fig. 5).The signal intensity emittedfrom the pH Sensitive LPHNPs (pHS-LPHNP-C6)was quantified higher than LPHNPs (LPHNP-C6) (Fig. 5), and results were attested by the quantification of DTX uptake through HPLC method. The increased cell uptake of pH sensitive LPHNPs is accredited with the cytosolic drug delivery [11,22,41] Our quantification results of cell uptake obtained with variousconcentrations are well supported by the qualitative observations of confocal laser scanning microscopy (CLSM). The cellular uptake results showed that drug uptake was not significantly (p > 0.05) proportionate to the concentration, and the greater uptake was seen at 40 µg/ml ( Fig. 6A, B). Our resultsshowing the internalisation of pHS-LPHNPs formulations were discovered time-dependent that leads to significant rise (p < 0.001) in DTX uptake with change of the incubation time from 1-2 hr [12] (

9%). Total injured cells (including early & late apoptosis and necrotic cells) with pHS-LPHNPs-DTX was
found to be higher (93%) than that seen with LPHNPs-DTX (78%) & free DTX (49%) (Fig. 7).These findings corroborate the results obtained withthe quantitative uptake assays, and suggest thebetter anticancer effect of pHS-LPHNPs-DTX formulation [36]. It happens due to the induction of greater rate of apoptosis of cancer cells

In vivo pharmacokinetics and bio-distributionstudy
Thepharmacokineticstudies were carried out to assess the effect of polymers and lipids on the circulation time of different LPHNP formulations. The release of DTX in the blood was assessed (Fig. 8A) and pharmacokinetic parameters were assessed upon administration of single dose in the experimentally-induced breast tumour mice model ( Table 2). Our findings confirm the increased circulation residence, improved half-life of DTX when loaded and delivered via pHS-LPHNPs and compared with free DTX formulations. The administered free DTX formulations were found in higher concentration in the circulation up-to 2 hr following are duction in drug concentration after 6 hr. Furthermore, LPHNPs-DTX and pHS-LPHNP-DTX formulations when administered through intravenous route showed low initial concentrations in blood, and then showed greater concentration of drug after 6hr of administration [11]. The decreasein the levels of DTX was seen from 6 to 72 hr. pHS-LPHNP formulations showed detectable serum DTX at the end of 72nd hr. The formulations of LPHNP showed 7-8 times higher mean residence time (MRT) of DTX than free DTX. Seven fold MRT was observed with LPHNP-DTX formulations and 8 times with pHS-LPHNPs-DTX formulations (Table 2).
Likewise, AUC total and half-life of LPHNPs-DTX was assessed higher than the free DTX (Table 2). Our results clearly showed the prolonged circulation time of pHS-LPHNP formulations and therefore suggesting the extended retention of DTX in the blood stream (Table 2). Consistentto our previous findings, results of this study advocate for the sustained and extended release behaviour of DTX when encapsulated into LPHNPs & pHS-LPHNPs [11].LPHNPs and pHS-LPHNPs showed greater plasma levels of DTX due to the double encirclement effect [19].
Therefore, these pharmacokinetic results suggest potential of pHS-LPHNPs for the effective and efficient delivery of DTX for developing treatment strategies against breast cancer. We believe these bio-  Estimation ofin vitro drug release from pHS-LPHNPs-DTX & LPHNPs-DTX at different pHs     The qualitative cell uptake study was carried out by using CLSM.

Figure 10
The qualitative cell uptake study was carried out by using CLSM.

Figure 11
The quantitative cell uptake study was carried out by the cell lysis method       Schematic for the development of DTX loaded pH Sensitive lipid polymer hybrid nanoparticles (pHS-LPHNPs-DTX)