In vitro potency, in vitro and in vivo efficacy of liposomal alendronate in combination with γδ T cell immunotherapy in mice

Nitrogen-containing bisphosphonates (N-BP), including zoledronic acid (ZOL) and alendronate (ALD), have been proposed as sensitisers in γδ T cell immunotherapy in pre-clinical and clinical studies. Therapeutic efficacy of N-BPs is hampered by their rapid renal excretion and high affinity for bone. Liposomal formulations of N-BP have been proposed to improve accumulation in solid tumours. Liposomal ALD (L-ALD) has been suggested as a suitable alternative to liposomal ZOL (L-ZOL), due to unexpected mice death experienced in pre-clinical studies with the latter. Only one study so far has proven the therapeutic efficacy of L-ALD, in combination with γδ T cell immunotherapy, after intraperitoneal administration of γδ T cell resulting in delayed growth of ovarian cancer in mice. This study aims to assess the in vitro efficacy of L-ALD, in combination with γδ T cell immunotherapy, in a range of cancerous cell lines, using L-ZOL as a comparator. The therapeutic efficacy was tested in a pseudo-metastatic lung mouse model, following intravenous injection of γδ T cell, L-ALD or the combination. In vivo biocompatibility and organ biodistribution studies of L-N-BPs were undertaken simultaneously. Higher concentrations of L-ALD (40–60 μM) than L-ZOL (3–10 μM) were required to produce a comparative reduction in cell viability in vitro, when used in combination with γδ T cells. Significant inhibition of tumour growth was observed after treatment with both L-ALD and γδ T cells in pseudo-metastatic lung melanoma tumour-bearing mice after tail vein injection of both treatments, suggesting that therapeutically relevant concentrations of L-ALD and γδ T cell could be achieved in the tumour sites, resulting in significant delay in tumour growth.

formed by removing the solvent under reduced pressure by a rotary evaporator. The lipid film was then re-dissolved in 3 ml diethyl ether and 3 ml chloroform. The aqueous phase (HBS, 1.5 ml) was then added and the solution was emulsified by sonicating for 10 min at 60°C in a bath-type sonicator (Ultrasonic Cleaner, VWR). The resulting emulsion was placed on the rotary evaporator to remove the organic solvent under reduced pressure. Evaporation continues until a gel is formed; further evaporation causes spontaneous formation of liposomes and subsequently ensures that all traces of the organic solvent have been removed.
A final volume of 1 ml liposome suspension was recovered and stored at 4°C.

Physicochemical characterisation of liposomes
The hydrodynamic diameter, polydispersity index and zeta potential of the liposomes were measured using the NanoZS (Malvern Instrument, UK). Hydrodynamic size, polydispersity index and Zeta potential were measured in disposable square polystyrene cuvettes and disposable capillary cells, respectively (Malvern Instrument, UK). The original sample (20 µl) was diluted to 1.5 ml with 10 mM sodium chloride. The measurements were carried out at 25C. Three measurements were performed and the mean and standard deviation were calculated for each sample.

Lipid recovery quantification
Stewart's assay was used to determine the lipid concentrations before and after purification with size exclusion chromatography [1]. Stewart's reagent (0.1 M ammonium ferrothiocyanate solution) was prepared using 5.46 g ferric chloride hexahydrate and 6.08 g of ammonium thiocyanate and made up to 200 ml with deionised water. The solution to be measured was prepared by mixing 50 μl of the unknown sample (or the standard), 2 ml of chloroform and 2 ml of Stewart's Reagent. The mixture was centrifuged at 1000 g in a bench centrifuge (Centrifuge 5810 R, Eppendorf) for 10 min and the organic layer was removed and analysed with UV spectrometer (Lambda 35, Perkin Elmer). The absorbance at 485 nm was used to determine the lipid concentration. Calibration curves were prepared in the same way using known amounts of lipid as standards. The lipid recovery was determined by comparing the lipid concentration of a liposome sample before and after purification.

Quantification of ZOL
Two different methods were used to quantify ZOL in liposomal formulations; Reverse Phase High Performance Liquid Chromatography (RP-HPLC) [2] with UV spectroscopy, or UV detection alone. UV spectroscopy alone was used to determine the percentage encapsulation efficiency (% EE) of the liposomes. RP-HPLC with UV spectroscopy was used in release studies since a method with greater sensitivity was required.

Sample processing and ZOL standard curves
A calibration curve containing 5 mM empty liposomes and known concentrations of free ZOL, referred to 'ZOL spiked liposomes' samples, were prepared. ZOL concentrations ranged between 0.1-1 mM and 40-400 µg/ml (2-20 µg per 50 µl injection volume) for samples quantified with UV spectroscopy or RP-HPLC, respectively. A calibration curve containing free ZOL at the same concentration range was prepared to ensure that the presence of lipid did not interfere with the measurements. L-ZOL samples to be quantified (or standards) were both processed using the Folch method prior to quantification. This method disrupts the liposomes; allowing the encapsulated ZOL to be released into the aqueous phase and separating any hydrophobic components (cholesterol, lipids etc.) from the hydrophilic drug. In brief, chloroform and methanol were added to a sample of liposomes at 8:4:3 (chloroform: methanol: liposome suspension) volume ratio. The sample was then vortexed (Vortex genie 2, Scientific Industries Inc, USA) and centrifuged at 10000 rpm for 10 minutes (Centrifuge 5810 R, Eppendorf). Two layers were formed after centrifugation and the upper aqueous layer containing the ZOL was removed and quantified using one of the two methods described below: UV spectroscopy A 0.5 ml sample of the upper aqueous phase was added to 0.5 ml of DI water. The samples were then read for absorbance at 210 nm with UV spectrometer (Lambda 35, Perkin Elmer), against a HBS reagent blank. Concentrations were calculated from the 'ZOL spiked liposomes' calibration curve.

RP-HPLC with UV spectroscopy
A 0.5 ml sample of the upper aqueous phase was transferred to an HPLC vial for analysis.
The Jasco HPLC instrument was used with a Jasco PU-2089 Plus pump, Jasco CO-2067 Plus oven, Hasco UV-2075 Plus UV/Vis Detector and Jasco AS-2050 Plus Sampler. The quantitative analysis of ZOL was performed on a Gemini C18 column (150x4.60 mm; 5 µ; 110 Å; Phenomenex UK). The mobile phase consisted of an aqueous buffer (8 mM dipotassium hydrogen orthophosphate, 2 mM di-sodium hydrogen orthophosphate and 7 mM tetra-n-butyl ammonium hydrogen sulphate adjusted to pH 7.0 with sodium hydroxide) and acetonitrile (85:15). The mobile phase was filtered through a 0.2 µm membrane filter and degassed by sonication (0.5 hr/L) before use. The flow rate was 1.0 ml/min with isocratic conditions used. The temperature of the column was 35°C. The wavelength of the UV/Vis detector was set at 210 nm. Concentrations were calculated from the 'ZOL spiked liposomes' calibration curve.

Quantification of ALD
ALD concentrations, for determination of % EE or percentage drug released, were determined with a copper sulphate-based UV spectroscopy method [3] or o-phthalaldehyde (OPA)-based fluorescence method [4], respectively.

Sample processing and ALD standard curves
A calibration curve containing 5 mM empty liposomes and known concentrations of free ALD, referred to 'ALD spiked liposomes' samples, was prepared. ALD concentrations ranged between 0.1-1 mM and 0.5-5 µM for samples quantified with copper sulphate-based UV detection method or o-phthalaldehyde (OPA)-based fluorescence method, respectively. L-ALD samples to be quantified (or standards) were both processed using the Folch method prior quantification as described above for ZOL. The upper aqueous layer containing the ALD was removed and quantified using one of the two methods described below: Quantification using copper sulphate Quantification using OPA/ 2ME reagent OPA/2ME reagent was prepared using 10 mg of OPA, 50 µL of 2ME, with the volume completed to 10 mL using 0.05 M NaOH. A 0.2 ml sample of the upper aqueous layer to be assessed for ALD was mixed with 0.1 mL of OPA/2ME reagent and the volume was completed to 2 ml with 0.05 M NaOH. The emission intensity was recorded between 380-600 nm at 360 nm excitation wavelength using a luminescence spectrophotometer (Perkin Elmer, Model: LS50B). The absorbance was read at 450 nm emission for all samples.
Concentrations were calculated from the 'ALD spiked liposomes' calibration curve.

Quantification of N-BP encapsulation efficiency (%EE) and drug loading
The drug loading and encapsulation efficiency was quantified with UV spectroscopy (ZOL), copper sulphate and UV spectroscopy (ALD) and Stewart's assay (lipid). The amount of drug entrapped within liposomes was quantified for this purpose. A sample of the liposomes was taken and processed using the Folch method, the N-BP in the upper aqueous layer was then quantified with RP-HPLC (210 nm) (ZOL) or OPA method (ALD), as described above.
Encapsulation Efficiency (EE %) was expressed as the percentage of N-BP loaded from the initial amount used, taking into account dilution factors. Drug loading was expressed as N-BP's µmol per lipid's µmol in the purified liposome sample. The quantity of N-BP in each liposome sample was measured three times and expressed as mean ± standard.

Liposome release studies
Drug release was carried out using the dialysis method [1]. One millilitre containing L-ZOL, L-ALD, or the free drug as controls (~5 µmol), was placed inside a 10 kD MWCO dialysis bag, in the presence or absence of 50% FBS, and dialysed against 200 ml HBS at 37 o C under sink condition. Samples were obtained from inside (50 µl, L-ZOL) or outside (3 ml, L-ALD) the dialysis bag at different time points (t = 0.25, 0.5, 1, 2, 4, 8 and 24 h) and replaced with fresh HBS. ZOL and ALD contents were assessed with RP-HPLC (210 nm) and OPA method, respectively, as described above. L-ZOL samples had to be taken from inside the dialysis bag due to sensitivity limits of the detection method, whereas in the case of L-ALD the detection method used was sufficiently sensitive to allow samples to be taken from outside the dialysis bag. Percentage release was quantified by measuring the change in ZOL or ALD concentration inside or outside the dialysis bag, respectively.
Results were expressed as mean ± standard. Each experiment was performed in triplicate. Glutamax and 1% antibiotic-antimycotic solution) at a concentration of 3x10 6 cells/ml. In order to expand the γδ T cells, the PBMCs were activated with 1 µg/ml ZOL and 100 U/ml IL-2. Additional medium and 100 U/ml IL-2 were added every 2-3 days for 15 days.

Flow cytometry analysis of γδ T cells
On Day 1 and Day 15, 200 µl samples of the cell suspension were taken and 5 µl of either T Cell Receptor (TCR) Pan γ/δ-FITC antibody or IgG1 FITC Isotype control antibody was added. The cells were incubated with the antibodies for 20 min at 4°C before 1 ml PBS was added. Cells were centrifuged at 1000 rpm for 5 min in a bench centrifuge (Centrifuge 5810 R, Eppendorf). The supernatant was discarded and the cell pellet was re-suspended in 500 µl of PBS. All flow cytometric data were acquired using a Beckman Coulter Cytometer FC 500 MPL and were analysed using CXP Analysis software (Beckmann Coulter). The lymphocyte cell population was gated and the number of cells in this gate that express the γδ TCR were calculated as a percentage of the total lymphocytes. were prepared using the TFH and RVE methods (Table S1).

Cell Viability Equation
To quantify the amount of ZOL and ALD encapsulated into liposomes, different quantification methods were developed, as described in the supplementary information. ZOL content was measured using UV-Vis and HPLC ( Figure S2), while ALD encapsulation efficiency was determined using copper sulphate-based UV spectroscopy method and ophthalaldehyde (OPA)-based fluorescence method ( Figure S3). Our results showed that both ZOL and ALD had similar encapsulation efficiencies (% EE) ranging from 5.2 -6.4%, with no significant differences between the TFH and RVE methods (Table S2). Drug loading of 0.23 -0.27 mmol ZOL or ALD per mmol lipid was obtained (p> 0.05). As both preparation methods resulted in similar outcomes, TFH was adopted to formulate liposomes for all subsequent experiments, since it is less-time consuming.

L-ZOL and L-ALD showed low drug release in the presence of serum
In order to predict the in vivo stability of the liposomal formulations, we evaluated the drug release of L-ZOL and L-ALD at 37°C in both HBS and 50%. L-ZOL or L-ALD (or free drugs as controls) were suspended in HBS or 50% FBS and these preparations were placed in a dialysis bag with a 10 kD molecular weight cut off (MWCO). Dialysis was performed against HBS while maintaining sink conditions. Free ZOL or ALD was shown to readily exit the dialysis bag, with over 97% release by 4 h in all conditions, indicating that drug release was not impeded by the dialysis bag or the presence of serum ( Figure S4).
In contrast to the free drugs, L-ZOL and L-ALD showed slower release profiles under similar conditions. In the absence of serum, L-ZOL and L-ALD showed release of ~ 2, 3 and 15% and 1, 5 and 5%, at 1, 8 and 24 h, respectively. In presence of serum, these values were ~ 2, 12 and 27% (p < 0.05) and 3, 11 (p< 0.05) and 17% (p< 0.05), respectively (FBS vs. HBS). It was concluded that there was a significant, but nonetheless slight (<10%) increase in drug release from the liposomes, when incubated with FBS. In light of the satisfactory stability of these formulations, we proceeded to in vitro or in vivo testing of their anti-tumour activity.

% EE and drug loading of ZOL in liposomes
A calibration curve of ZOL, in the range 0.1-1 mM, was achieved using the UV method, with no interference from liposomal components observed. This method allowed for quick and simple measurement of ZOL and was sensitive enough for determination of ZOL % EE in L-ZOL. However, for drug release studies, a more sensitive method was required. RP-HPLC was able to measure ZOL at concentrations as low as 40 µg/ml. Chromatograms of empty liposomes, ZOL, ZOL spiked liposomes and L-ZOL are shown in Figure S2A. ZOL showed retention times of 6 minutes, and no interference from the lipid was experienced.
Additionally, ZOL spiked liposomes or ZOL encapsulated liposomes (ZOL) produced a peak at 6 minutes, matching the elution profile of free ZOL. A calibration curve using ZOL spiked liposomes was prepared, and was used for quantification of ZOL concentration in L-ZOL ( Figure S2B).

% EE and drug loading of ALD in liposomes
Calibration curves for ALD were obtained in the range 0.1-1 mM and 0.5-5 µM using copper sulphate-based UV detection method or OPA-based fluorescence method, respectively ( Figure S3). Linear relationships between ALD concentrations and UV absorbance or fluorescence intensities were observed. No interference from residual lipids or other reagents used in the sample preparation process was seen. The copper sulphate-based UV detection method was sufficiently sensitive to quantify ALD % EE in L-ALD but its limit of detection was above that required for drug release studies. The OPA-based fluorescence method was ~200 times more sensitive and was used to quantify ALD in drug release studies.

γδ T cells isolated and purity quantified following expansion
γδ T cells were obtained from whole blood from healthy volunteers (n=22). γδ T cells were selectively expanded from the peripheral blood mononuclear cells (PBMC) by addition of IL-2 and ZOL ( Figure S8). The percentage and number of γδ T cells increased dramatically over a 15 day period. On day 1, the donors had a γδ T cell population of 4.9 ± 5.9 % of their total lymphocyte fraction (0.8 -28.3 %) and by day 15 this had increased to 86.0 ± 9.3 % ( Figure   S9). The number of γδ T cells also increased with approximately 96 fold expansion in cell number between day 1 and day 15.  [a] Hydrodynamic diameter measured with dynamic light scattering.

SUPPLEMENTARY TABLES AND FIGURES
[b] Analysed with electrophoretic light scattering using 10 mM NaCl.
[c] Data are represented as mean ± SD (n=3).         The UV spectra and calibration curves obtained in the range 0.1 -1 mM using Cu complexation method. UV spectroscopy was used to quantify the ALD content of liposomes, whereby ALD was complexed with copper. ALD was released from the liposomes using the Folch method and the aqueous layer was removed and added to an equal volume of 10 mM copper sulphate. The UV absorbance at 240 nm was then measured. (B) The spectra and calibration curves obtained in the range 1 -5 µM using OPA fluorescence method. ALD was detected using fluorescence by conjugating ALD to o-phthalaldehyde (OPA) in order to quantify ALD released from the liposomes. The OPA reagent was prepared by using 10 mg of OPA, 50 µL of 2mercaptoethanol, with the volume completed to 10 mL using 0.05 M NaOH. The sample was excited at 360 nm and the emission was measured at 450 nm. Blood samples were obtained from healthy volunteers. The blood sample was layered on top of Ficoll-Paque Plus and centrifuged in order to isolate the peripheral blood mononuclear cells (PRMCs). The PBMCs were washed in PBS and then re-suspended in complete media at a concentration of 3x10 6 cells/ml. In order to preferentially expand the γδ T cells, the PBMCs were activated with 1 µg/ml ZOL and 100 U/ml IL-2. Additional medium and 100 U/ml IL-2 were added every 2-3 days for 15 days.

Red Blood Cells
Plasma PBMC Cells cultured in media with ZOL and IL-2 for 1 day.

Centrifugation
Expanded γδ T cell culture are obtained on day 15.  h. An MTT assay was then performed to assess cell viability. MTT solution was prepared in PBS at a concentration of 5 mg/ml and was diluted in media (1:6) prior to use. The supernatant of each well was removed and MTT solution was added to each well. The plates were then incubated at 37°C for 3 h before the MTT solution was removed from each well and replaced with DMSO solubilise the crystals that had formed. The absorbance was then read at 570 nm with reference at 630 nm. ). The co-culture was left for 24 h, followed by γδ T cells and cell washing prior MTT assay. These concentrations were chosen to reflect the amount of liposomes used when treating the cells with L-ZOL or L-ALD. No significant cytotoxicity was observed in the case of the empty liposomes alone or in combination with the γδ T cells, except for the cell line A375Pβ6 where a reduction in cell viability was seen at the highest concentration of liposomes used. Data are expressed as mean ± SD (n=5). p < 0.05, vs. (Student's t test vs. naïve).