Study Design
In this study, we established a systematic strategy for the synthesis of docetaxel-HSP70 (DHSP) conjugate and explored its antitumor potential. Here, we have targeted free -NH2 groups present on the surface of HSP70 for conjugation. The HSP70 was extracted from cancer cells using size exclusion chromatography and purified using immunoprecipitation with HSP70 specific monoclonal antibodies. Additionally, a cleavable ester bond with the carboxyl group (-COOH) was synthesized on docetaxel to conjugate it with the primary amines (-NH2) on HSP70 while sustaining the structural integrity of docetaxel as well as HSP70. HSP70 purity was assessed using proteomic characterization, while the docetaxel and final conjugate were assessed using FTIR and NMR spectrum analyses. The conjugate stoichiometric coefficient was determined using spectrophotometer and docetaxel release profile of conjugate was examined in vitro in four different buffers mimicking human philological conditions. Anticancer efficiency of docetaxel alone and in conjugation was evaluated on squamous cell carcinoma cancer.
Materials
Docetaxel (catalogue- 114977-28-5) was purchased from TCI Chemicals Pvt. Ltd (India), Sephadex G75 (Catalogue- G75120-50G), Phorbol-12-myristate-13-acetate (PMA) were purchased from Sigma Aldrich (USA). Anhydrous DMSO, diethyl ether, acetonitrile, pyridine, Glutaric anhydride, N-hydroxy succinimide (NHS), Ethyl-dimethyl-aminopropyl-carbodiimide (EDC), 4-(dimethylamino)-pyridine (DMAP), acrylamide, ammonium persulfate, isopropanol, protease inhibitor cocktail, N, N’-methylene-bis-acrylamide, sodium dodecyl sulfate, β-mcaptoethanol, Nile-RED dyewere purchased from SISCO Research Laboratories (Mumbai, India), centrifuge filter device with an MWCO of 10 kDa from (Millipore Billerica, Massachusetts). Membrane filters of 0.22 µm pore size, nitrocellulose membrane of 0.45 µm pore size, centrifugal devices with 10k and 20k MWCO exclusion limit were obtained from AXIVA (Sonipat Haryana INDIA), BioRad (Hercules, California, USA), and Pall Corporation (New York, USA). Dulbecco’s MEM (DMEM), Dulbecco’s MEM/Ham's F-12(1:1), Roswell Park Memorial Institute medium (RPMI 1640), Fetal bovine serum (FBS), antibiotic-antimycotic were purchased from Gibco BRL (Gaithersburg, MD, USA). Anti-human HSP70 (Catalogue-MA3-008), HRP-Goat anti-Mouse IgG (Catalogue-62-6820) and TRITC- Anti-Mouse IgG (H+L) (Catalogue-A16071). Dyes CalcineAM (Catalogue-13529), Mitospy (Catalogue-50-207-9911), DAPI (catalogue-62247) and apoptosis kit (catalogue-422201) were purchased from Biolegend (San Diego, USA) all other chemicals used in this study were of Analytical AR/Excel AR graded and were purchased from Merck Laboratories (Rahway, New Jersey, USA), SISCO Research Laboratories (Mumbai, India), and HiMedia Laboratories Private (Mumbai, India).
Methodology
Cell lines and culture
This study was carried out at the All India Institute of Medical Science using following cell lines: Oral squamous cell carcinoma (OSCC) CAL33 was gifted by Dr. RP Singh (JNU, New Delhi, India) and SCC4 was gifted by Dr. S.S. Chauhan (AIIMS, New Delhi, India). All cells were kept in the proper cell culture media, such as DMEM or RPMI1640, at 37 °C, 95% relative humidity, and 5% v/v CO2. Every 2 to 3 days, cells were passaged and used in the exponential growth phase and trypan blue dye exclusion were used to assess cell viability prior uses (>95%).
Heat Induced Overexpression of HSP70
OSCC cell lines SCC4 and CAL33 were cultured in complete DMEM/F12 supplemented with 10% FBS media at 37 °C. At 50-60% confluence old media was replaced with fresh complete DMEM/F12 media and cells were incubated at 42°C CO2 incubator for 1hr. At completion of 1hr, the cells were removed from 42 °C incubator and incubated in 37 °C CO2 incubator for 15 min to induce heat shock and overexpression of HSP70. Post completion of 15 min at 37 °C one set of cells were processed for ICC, qPCR and protein isolation, and remaining set were again incubating in 42 °C CO2 incubator, this cycle was repeated for six times to collect cells at different heat shock time interval of 1hr, 2hr, 3hr, 4hr, 5hr and 6hr as shown in Figure1A.
Confocal Laser Scan Microscopy
The cells grown on sterile round coverslip were fixed in 4% formalin for 10 minutes and incubated in 1% BSA solution for 20 min to reduce non-specific binding of antibodies. Cells on coverslip were incubated overnight at 4ºC in HSP70 primary antibody (1:1000) diluted in 1% BSA. Next day cells were counter-stained with TRITC labelled secondary antibody diluted in 1% BSA and incubated at RT for 2 hours in the dark. Following the incubation, cells were permeabilized with 0.1% Triton X-100 for 5 minutes and nucleus of cells were stained with DAPI (1 µg/ml) for 5 minutes at RT. Each step was followed by three washes of 5 minutes each with PBS. The coverslips were mounted in glycerol based mounting media with DABCO as quenching agent for microscope visualization (ECLIPSE 80I, Nikon Instruments Inc., NY, USA).
Quantitative Real-Time PCR
RNAiso Plus (Takara Bio USA) was used to isolate total RNA from cells. cDNA was synthesized from one microgram of RNA using the iScript™ cDNA Synthesis Kit. Real-time quantitative PCR reactions for Homo sapiens HSP70 (NM_005345.6) primers: Forward 5′ -AGGACATCAGCCAGAACAAG- 3′ and Reverse 5′ -CTGGTGATGGACGTAGAAG- 3′ were performed on the AriaMx Real-time PCR System (Agilent) as per recommended protocol using the Brilliant II SYBR® Green qPCR Master Mix (Agilent). GAPDH was used as an internal reference and each sample were set up in triplicate.
Western Blotting
The cell pellet was re-suspended in NP40 lysis buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% sodium dodecyl sulfate) with 1X protease inhibitor cocktails and incubated on ice for 30 minutes before undergoing a freeze-thaw cycle (freeze at -70 °C and thaw at 37 °C). The cell lysate was collected by centrifugation at 13,000 rpm for 15 min at 4 °C, followed by supernatant collection and the total protein estimation by BCA method. Total 30μg protein was mixed with 5 × SDS loading buffer and denatured by boiling at 100 °C for 5 min followed by SDS-PAGE with a stacking gel (4%) and separation gel (10%). Following SDS-PAGE proteins were transferred to nitrocellulose membrane. For western blotting membranes were incubated with the primary antibody at 4 °C overnight and 2 hours with HRP conjugated secondary antibody at room temperature. Each step was followed by three washes of 10 minutes each with TBS-t (0.1%). Blots were incubated in ECL for three minutes, and then they were exposed to a maximum of one minutes in the Aruas or BioRad Gel Doc system to detect chemiluminescence.
Size Exclusion Chromatography
To separate out proteins of 65-75 kDa molecular weight Sephadex® G-75 gel filtration chromatography was used. Sephadex® G-75 powder was swelled in 30 mM Tris (8.0 ph.) for 24hr. Once swelled the slurry was washed extensively with five volumes of 30 mM Tris (8.0 ph.) buffer and transferred to glass chromatography columns of 30 cm height and allowed for settling. Pre-cleansing of lysate was done by adding 1.0 μg/ml of the control IgG (normal mouse, anti-human IgG) cross-linked to magnetic beads. Total 1 ml of cell lysate (3 mg protein) was applied to the Sephadex® G-75 column. After the lysate was absorbed in Sephadex bed 30 ml of 30 mM Tris (8.0 ph.) elution buffer was applied for elution of different molecular weight proteins as shown in Figure 1A. Total 30 elutes of 1 ml each were collected and 30µl of each elute was used for protein quantitation using UV-visible spectrophotometer at 280 nm. Elutes having absorbance more than elution buffers were selected for BCA assay to determine protein concentration and protein in each elute was interpolated from BSA standard curve using Graph pad prism 9.31. The elutes having presence of protein were analyzed on SDS-PAGE gel for HSP70 presence and western blotting were performed with monoclonal HSP70 antibodies and pure HSP70 was taken as positive control along with β–actin as a loading control.
Immunoprecipitation of HSP70
Sephadex® G-75 gel has a lower and upper exclusion limit of 60 kDa and 65 kDa. In order to obtain pure HSP70, immunoprecipitation was done with the monoclonal HSP70 antibodies covalently cross-linked with the Protein-L agarose beads. Sephadex® G-75 elutes having presence of HSP70 were collected and incubated with HSP70AB-Protein-L beads at 4° C for 4hr. At completion of 4hr HSP70AB-Protein-L beads were collected by centrifugation at 2,500 rpm for 5 minutes at 4° C. Beads pellet were washed four times with 1.0 ml NP40 lysis buffer with protease inhibitors. After the final wash, beads pellets with HSP70 were processed for HSP70 elution from beads. For HSP70 elution, the beads were incubated at 56 °C in 50 µl of 1M Glycine buffer, pH 2.5 and reaction was terminated (at different time interval of- 10 min, 20 min, 30 min) by addition of 200 µl of 20 mM tris buffer, pH 10 neutralization buffer, followed by centrifugation at 12000 rpm for 5 minutes at 4 °C. Post centrifugation the supernatant was collected and beads were suspended in lysis buffer for western blotting to confirm elution of HSP70 from HSP70-beads complex and antibody IgG was used as isotype control. Glycine is a non-polar, glucogenic, non-optical, and nonessential amino acid with free carboxyl group (–COOH) at one end and primary amine group (-NH2) on another end. To remove free glycine and tris the immunoprecipitated HSP70 was diluted five times with 1X phosphate buffer and transitioned to a centrifuge filter tube and centrifuged against membrane with MWCO of 10 kDa (Millipore Billerica, Massachusetts), at 10000 rpm for 10 min, the filtrate was discarded, concentrated sample was collected and used for conjugation.
Synthesis of HSP70-Docetaxel Conjugate
HSP70 1A is 68-72 kDa protein (NCBI Reference sequence: NP_005337.2) histidine (His, H), Lysine (Lys, K), and arginine (Arg, R) are three positively charged polar amino acids. Lysine and arginine both have two primary amine groups, one positively charged ε-primary amino group as a side chain, with higher pKa (Lysine pKa ~10.5 and Arginine pKa ~12) and other α-primary amino group at N-terminus. The side chains in lysine are highly reactive and involved in forming hydrogen bonds with negatively charged non-protein atoms (e.g., anions or carboxylate groups). In this study we targeted free NH2- amine groups present on the HSP70 protein surface to conjugate with docetaxel (MW 807.879 g/mol). Synthesis of bio-conjugate of docetaxel and HSP70 was carried out in two steps- first synthesizing carboxyl functional groups (–COOH) on docetaxel, second conjugating docetaxel-COOH (docetaxel-glutarate) with HSP70 primary amino (-NH2) group of lysine, its proposed mechanism is shown in Figure 1B-C.
Synthesizing Carboxyl Functional Group (–COOH) on Docetaxel
In order to conjugate docetaxel with the primary amino (R-NH2) group of lysine in HSP70, carboxyl functional group (–COOH) is required and docetaxel do not have free carboxyl functional groups. To create a functional carboxyl group on docetaxel, hydroxyl (-OH) functional group present on carbon 10 in docetaxel was targeted(19). Initially, docetaxel-glutarate was synthesized, briefly, equvmolar concentration of- docetaxel, glutaric anhydride, and pyridine was dissolved in 5 ml DMSO (as solvent) and stirred for 48hr at room temperature to form docetaxel-glutarate (docetaxel-COOH). After 48 hrs reaction solution was washed three times with 50 ml of deionized water: ethyl acetate, to remove unreacted glutaric anhydride, pyridine and the organic phase (ethyl acetate) with docetaxel-glutarate (docetaxel-COOH) was collected and lyophilized for FTIR and 1 H-NMR characterization as shown in Figure 1B (20).
Conjugation of Docetaxel-COOH with HSP70-NH2
For conjugating docetaxel-COOH with NH2-HSP70, the HSP70 was incubation with the 50 units of β-mercapranol in 1.8 ml of pH8.1 0.1 M NaHCO3 buffer and mixed gently for 5 min at room temperature, followed by addition of 0.1% ammonia to stop the β-mercapranol activity. For covalent conjugation of carboxyl (–COOH) group with primary amine group (-NH2), the–COOH in needs to be activated. For activating–COOH equvmolar concentration of lyophilized docetaxel-glutarate, EDC, NHS, and DMAP in 1 ml DMSO (as a solvent) were mixed and stirred for 20 min at room temperature (21). To the0.4 µm HSP70 mixture 24 µm activated docetaxel-glutarate was added dropwise and the reaction was left to proceed for 6hr at room temperature followed by 16hr at 4 °C. At completion of reaction, whole reaction solution was collected and centrifuged at 10000 rpm against membrane with MWCO of 10 kDa to remove any free unconjugated docetaxel-glutarate and other salts as shown in Figure 1B. The resulting filtrate and concentrated sample were collected and analysed by FTIR, western blotting and NMR, to confirm the conjugation and to determine psychometric ratio of HSP70: Docetaxel in the final DHSP conjugate as illustrated in Figure1C.
Fourier transform infrared spectroscopy (FTIR)
Fourier transform infrared spectroscopy (FTIR) was used for the analysis of functional groups on the synthesized compound. For analysis 10 µl of the sample was used and data were recorded using Agilent Cary 670 benchtop FTIR-spectrometer (Agilent Technologies, Inc. Santa Clara, California, USA) with two different ATRs -Microdiamond A.T.R. and Microge A.T.R. and wave number range of 4000 cm-1 to 400 cm-1 were recorded.
Nuclear Magnetic Resonance (NMR)Spectroscopy
The 1H NMR spectra were measured with on Varian INOVA 50 NMR Spectrometer (Agilent Technologies, Inc. Santa Clara, California, USA). All the samples were completely dissolved in deuterated and centrifuged at 12000rmp to remove any solid remnants. For spectra measurement of docetaxel and docetaxel-glutarate samples were prepared in deuterated DMSO-d6 (C2D6OS) and spectra at 500 MHz were recorded. For HSP70 and HSP70-Docetaxel spectrum measurement samples were prepared in Heavy Water (D2O). NOESY Pulse sequence was used to acquire protein (HSP70 and HSP70-Docetaxel) 2D 1H NMR spectra.
Drug binding efficiency & Psychometric ratio calculation
For determining the UV absorbance wavelength, the docetaxel was dissolved in PBS and DMSO at concentration of 100 µg/ml and absorbance spectrum were scanned from 200 nm to 900 nm. The wavelength with the highest absorbance 377.6nm was recorded, and the standard curve plot was generated in GraphPad prism from serially diluting (500 µg to 30 ng) docetaxel in respected medium. For determining psychometric ratio of HSP70: Docetaxel in the final conjugate, the 10 kDa filtrate membrane elute with unbounded docetaxel and concentrated samples with Docetaxel-HSP70 were taken and absorbance at 377.6nm was recorded and concentration was determined from plotted standard curve(22).The following equation was used to calculate drug binding efficiency (23):
In vitro Drug release assay
Two approaches were used to determine the drug release from conjugation. In the first approach, to imitate the endosomal, tutorial, serum and plasma conditions, a drug release study was carried out by employing the dialysis technique in three distinct media with pHs of 5.5, 6.5 (cancer cell conditioned media), and 7.00, respectively. Briefly, 100ul of an HSP-Docetaxel conjugate with were placed into a tube with cellulose dialysis membrane filtration devices (cut-offs 12–14 kDa), and submerged in 10 ml of respected release medium and incubated at 37 °C. One millilitre of the released media was collected out of the released media at intervals of 0, 1, 2, 4, 6, 12, 24, 48 and 72 hours and replaced with the same amount of fresh medium as shown in Figure 1D. Collected one millilitre of the release media was evaluated for released docetaxel in a spectrophotometer. In the second approach, pure drug and HSP70 loaded with docetaxel were added to media at a ratio of 1:9 and incubated at 37 °C. One millilitre of the release medium was withdrawn from the released media at intervals of 0, 0.5, 1, 2, 4, 6, 12, 24, 48 and 72 hours (23). The samples were transitioned to a centrifuge filter device with an MWCO of 10 kDa from, and centrifuged at 9000 g for 10 min and filtrates were collected, diluted in ethanol (1:2 v/v) and analysed with spectrophotometer. In order to determine the amount of docetaxel released from the HSP-Docetaxel conjugate, the data were subsequently multiplied by the dilution factor. Readings at 377.6 nm were recorded and compared with standard curves to estimate released amount of docetaxel.
Cytotoxicity Assay
Cells in the exponential growth phase were seeded into each well of a 96-well plate at the density of 5 × 103 cells per well in 100μl volume, with three wells for each set of conditions. Cells were exposed to docetaxel, HSP70, and DHSP at different concentrations for 24hr, 48hr, 72hr, 96hr and 120 hr. Then 10μl of 5 mg/ml MTT was added to each well and incubated at 37 °C incubator for 4 hr followed by addition of 100μl of DMSO in each well to dissolve the formazan crystals. Biotek microplate reader was used to measure the optical density (OD) at a wavelength of 570 nm. Percent cell viability in each well was calculated using the following equation:
GraphPad prism software was utilized to plot the percent cell viability versus drug concentration curve and to calculate inhibitory concentration 50 (IC50).
FITC labelling of DHSP
HSP-Docetaxel was labelled with fluorescein isothiocyanate (FITC) for cellular experiments. As a representative example, 2 mg of DHSP was dissolved in 1 ml of 0.1 M NaHCO3 buffer pH8.1, and 0.1 mg of FITC solution (2 mg/ml in DMF) was added (24). The reaction mixture was stirred at RT for 6hr followed by stirring at 4 °C overnight. To remove excess unbounded FITC whole reaction solution was washed with NaHCO3 buffer three times by centrifuged at 10000 RPM against membrane with MWCO of 10 kDa (Millipore Billerica, Massachusetts) to concentrate FITC labeled DHSP. The resulting filtrate was discarded and concentrated sample was collected and analyzed by spectrophotometer to confirm FITC labeling of conjugate. All steps were performed under dark conditions. To determine the degree of labeling following equation was used:
Moles dye per mole protein = [Amax of labelled protein ÷ (ε X protein concentration (M))] X DF
Intracellular DHSP localization
The SCC4 and CAL33cells were grown on sterile coverslip in six well plates. At 60-70% confluence the cells were stained with lipid staining Nile-RED dye for 10 min, followed by washing and treatment with IC50 dose of FITC labelled Docetaxel-HSP70 (DHSP) conjugate and at different time interval- 0hr, 15 min, 30 min,1hr, 2hr, 4hr 8hr, 16hr, 24hr, 48 hr& 72hr single coverslip were removed and reaction was terminated by fixing the cells in 4% PFA for 2 min followed by staining cells with DAPI and mounting in glycerol based mounting media. All steps were performed under dark condition and each step was followed by washing coverslip with PBS three times with PBS with incubation of 5 min each.
Cell Cycle Analysis
Cells were incubated in incomplete DMEM media for 24hrs post treatment with IC50 concentration of docetaxel and DHSP for different time interval. Post 72hrs of treatment the cells were trypsinzed and fixed by dropwise addition of 1-2 ml of precooled 70% ethanol and incubated at -20 °C overnight. Next day cells were suspended in 500μl of RNase A (100 u/ml) containing PBS buffers and incubated at 37 °C for 30 min, followed by addition of 10ul PI (2 mg/ml) and incubation in dark for 30 min. Flow cytometry was performed in BD LSR Fortessa and the cell cycle analysis was done using FlowJo software.
Apoptosis Assay
Apoptosis was detected with annexinV/PI Double-Dye Apoptosis Kit. The cells treated with IC50 concentration of docetaxel and DHSP for 72hrwere trypsinzed with 0.05% trypsin to obtain a single-cell suspension, and centrifuged in 1500 rpm for 3 min. The cell pallet was dissolved in 500μl of binding buffersand stained with 5μl of AnnexinV-FITC and 5μl of PI according to the supplier’s directions. Each step was followed by three washes of 5 min each with PBS.
CalcineAM retaining assay.
Cells treated with IC50 concentration of docetaxel and DHSP for 72hr were washed with incomplete DMEM/F12 media and stained with 10 nm/ml of CalcineAM for 15 min at 37 °C followed by washing. Post staining with CalcineAM the cells were incubated for 4hrs in Co2 incubators and trypsinzed with 0.05% trypsin to obtain a single cell suspension, and stained with 10 µl of PI/DAPI (1 mg/ml) in dark for 5min. The cells were washed, and cell pellets were re-suspended in PBS buffer for flow cytometry analysis.
Mitochondrial Mass Assay
Cells treated with IC50 concentration of docetaxel and DHSP for 72hr were trypsinized with 0.05% trypsin and stained with 100nM MitoSpy™ GreenFM in incomplete DMEM/F12 culture media for 30 min at 37 °C followed by MitoSpy inactivation by adding 5 ml of complete DMEM/F12 media and stained with 10 µl of PI/DAPI (1 mg/ml) in dark for 5min. The cells were washed and cell pellets were re-suspended in PBS buffer for flow cytometry analysis.
Statistical analysis
All experiments were independently repeated three times, and the results are expressed as the mean ± S.D. The statistical significance was analysed using one-way ANOVA with Tukey’s multiple comparisons test to compare the differences between the experimental group and the control group. The data were considered significantly different at a p value < 0.05. The data and graphs were generated using the GraphPad Prism 8.00 software.