Intracellular Activation of a Prostate Specific Antigen-Cleavable Doxorubicin Prodrug: A Key Feature Toward Prodrug-Nanomedicine Design.

L-377,202 prodrug (Dox-PSA) was in phase I clinical trials for patients with metastatic castration-resistant prostate cancer (mCRPC). It consists of doxorubicin (Dox) conjugated to a prostate specific antigen (PSA)-cleavable peptide that can be selectively activated by secreted PSA at the tumor site. However, despite the initial promising results, further clinical testing with Dox-PSA was halted due to toxicity concerns emerging from non-PSA-specific cleavage, following systemic administration. In the present study, we have reported, for the first time, the intracellular activation of Dox-PSA, where Dox nuclear uptake was specific to C4-2B (PSA-expressing) cells, which agreed with the cytotoxicity studies. This finding was confirmed by encapsulating Dox-PSA prodrug into pH-sensitive liposomes to enable prodrug intracellular release, followed by its enzymatic activation. Interestingly, our results demonstrated that Dox-PSA loaded into pH-responsive nanoparticles exhibited cytotoxicity comparable to free prodrug in C4-2B monolayers, with superior activity in tumor spheroids, due to deeper penetration within tumor spheroids. Our approach could open the doors for novel Dox-PSA nanomedicines with higher safety and efficacy to treat advanced and metastatic prostate cancer.

Passively Targeted Curcumin-Loaded PEGylated PLGA Nanocapsules for Colon Cancer Therapy In Vivo.

Clinical applications of curcumin for the treatment of cancer and other chronic diseases have been mainly hindered by its short biological half-life and poor water solubility. Nanotechnology-based drug delivery systems have the potential to enhance the efficacy of poorly soluble drugs for systemic delivery. This study proposes the use of poly(lactic-co-glycolic acid) (PLGA)-based polymeric oil-cored nanocapsules (NCs) for curcumin loading and delivery to colon cancer in mice after systemic injection. Formulations of different oil compositions are prepared and characterized for their curcumin loading, physico-chemical properties, and shelf-life stability. The results indicate that castor oil-cored PLGA-based NC achieves high drug loading efficiency (≈18% w(drug)/w(polymer)%) compared to previously reported NCs. Curcumin-loaded NCs internalize more efficiently in CT26 cells than the free drug, and exert therapeutic activity in vitro, leading to apoptosis and blocking the cell cycle. In addition, the formulated NC exhibits an extended blood circulation profile compared to the non-PEGylated NC, and accumulates in the subcutaneous CT26-tumors in mice, after systemic administration. The results are confirmed by optical and single photon emission computed tomography/computed tomography (SPECT/CT) imaging. In vivo growth delay studies are performed, and significantly smaller tumor volumes are achieved compared to empty NC injected animals. This study shows the great potential of the formulated NC for treating colon cancer.

Cationic Liposome- Multi-Walled Carbon Nanotubes Hybrids for Dual siPLK1 and Doxorubicin Delivery In Vitro.

PURPOSE: To formulate f-MWNTs-cationic liposome hybrids for the simultaneous delivery of siPLK1 and doxorubicin to cancer cells. METHOD: f-MWNTs-cationic liposome hybrids were prepared by the thin film hydration method where the lipid film was hydrated with 100 µg/ml or 1 mg/ml of ox-MWNTs-NH3 (+) or MWNTs-NH3 (+) in 5% dextrose. siRNA complexation and protection ability was determined by agarose gel electrophoresis. f-MWNTs and liposome interaction was evaluated using Nile Red (NR) fluorescence spectroscopy. Cellular uptake in A549 cells was assessed by flow cytometry. Silencing of target proteins was determined by Luciferase and MTT assays. Sub-G1 analysis was performed to evaluate apoptosis following co-delivery of siPLK1 and Doxorubicin (Dox). RESULTS: Zeta potential and siRNA complexation profile obtained for all hybrids were comparable to those achieved with cationic liposomes. ox-MWNTs-NH3 (+) showed greater extent of interaction with cationic liposomes compared to MWNTs-NH3 (+). ox-MWNTs-NH3 (+) was able to protect siRNA from nuclease-mediated degradation. Enhanced cellular uptake of both the carrier and loaded siRNA in A549 cell, were observed for this hybrid compared to the liposomal carrier. A synergistic pro-apoptotic effect was obtained when siPLK1 silencing was combined with doxorubicin treatment for the hybrid:siRNA complexes compared to the lipoplexes, in A549 cells in vitro. CONCLUSIONS: f-MWNTs-cationic liposome hybrid designed in this study can serve as a potential vehicle for the co-delivery of siRNA and cytotoxic drugs to cancer cells in vitro.

Liposome-gold nanorod hybrids for high-resolution visualization deep in tissues.

The design of liposome-nanoparticle hybrids offers a rich toolbox for the fabrication of multifunctional modalities. A self-assembled liposome-gold nanorod hybrid vesicular system that consists of lipid-bilayer-associated gold nanorods designed to allow deep tissue detection, therapy, and monitoring in living animals using multispectral optoacoustic tomography has been fabricated and characterized in vitro and in vivo.

Liposomes: from a clinically established drug delivery system to a nanoparticle platform for theranostic nanomedicine.

For decades, clinicians have used liposomes, self-assembled lipid vesicles, as nanoscale systems to deliver encapsulated anthracycline molecules for cancer treatment. The more recent proposition to combine liposomes with nanoparticles remains at the preclinical development stages; however, such hybrid constructs present great opportunities to engineer theranostic nanoscale delivery systems, which can combine simultaneous therapeutic and imaging functions. Many novel nanoparticles of varying chemical compositions are being developed in nanotechnology laboratories, but further chemical modification is often required to make these structures compatible with the biological milieu in vitro and in vivo. Such nanoparticles have shown promise as diagnostic and therapeutic tools and generally offer a large surface area that allows covalent and non-covalent surface functionalization with hydrophilic polymers, therapeutic moieties, and targeting ligands. In most cases, such surface manipulation diminishes the theranostic properties of nanoparticles and makes them less stable. From our perspective, liposomes offer structural features that can make nanoparticles biocompatible and present a clinically proven, versatile platform for further enhancement of the pharmacological and diagnostic efficacy of nanoparticles. In this Account, we describe two examples of liposome-nanoparticle hybrids developed as theranostics: liposome-quantum dot hybrids loaded with a cytotoxic drug (doxorubicin) and artificially enveloped adenoviruses. We incorporated quantum dots into lipid bilayers, which rendered them dispersible in physiological conditions. This overall vesicular structure allowed them to be loaded with doxorubicin molecules. These structures exhibited cytotoxic activity and labeled cells both in vitro and in vivo. In an alternative design, lipid bilayers assembled around non-enveloped viral nanoparticles and altered their infection tropism in vitro and in vivo with no chemical or genetic capsid modifications. Overall, we have attempted to illustrate how alternative strategies to incorporate nanoparticles into liposomal nanostructures can overcome some of the shortcomings of nanoparticles. Such hybrid structures could offer diagnostic and therapeutic combinations suitable for biomedical and even clinical applications.

Mild hyperthermia accelerates doxorubicin clearance from tumour-extravasated temperature-sensitive liposomes.

Mild hyperthermia (HT) (40-43 °C) has been combined with temperature-sensitive liposomes (TSL), offering on-demand drug release for increased drug bioavailability and reduced systemic toxicity. Different HT regimens have been applied to trigger liposome drug release in the blood vessels (intravascular) of heated tumours or following tumour extravasation (interstitial). The present study systematically assessed the in vivo doxorubicin (Dox) release and therapeutic efficacy of Dox-loaded TSL with different release profiles. Low temperature-sensitive liposomes (LTSL-Dox), traditional-temperature-sensitive liposomes (TTSL-Dox), and non-temperature-sensitive liposomes (NTSL-Dox) were combined with a single or two HT in different tumour models (murine melanoma B16F10 tumour and human breast MDA-MB-435). The efficacy of each treatment was assessed by monitor tumour growth and mice survival. The level of Dox in tumour tissues was quantified using 14C-Dox and liquid scintillation while Dox release was assessed using live imaging and confocal laser scanning microscopy. Applying a second HT to release Dox from extravasated TTSL-Dox was not therapeutically superior to single HT application due to Dox clearance from the extravasated TTSL-Dox. Our findings revealed that enhanced blood perfusion in heated tumours during the second water bath HT could be seen as a hurdle for TTSL-Dox's anticancer efficacy, where the systemic toxicity of the redistributed Dox from the tumour tissues could be potentiated.

Encapsulation of doxorubicin prodrug in heat-triggered liposomes overcomes off-target activation for advanced prostate cancer therapy.

L-377,202 prodrug consists of doxorubicin (Dox) conjugated to a prostate-specific antigen (PSA) peptide substrate that can be cleaved by enzymatically active PSA at the tumor site. Despite the initial promise in phase I trial, further testing of L-377,202 (herein called Dox-PSA) was ceased due to some degree of non-specific activation and toxicity concerns. To improve safety of Dox-PSA, we encapsulated it into low temperature-sensitive liposomes (LTSL) to bypass systemic activation, while maintaining its biological activity upon controlled release in response to mild hyperthermia (HT). A time-dependent accumulation of activated prodrug in the nuclei of PSA-expressing cells exposed to mild HT was observed, showing that Dox-PSA was efficiently released from the LTSL, cleaved by PSA and entering the cell nucleus as free Dox. Furthermore, we have shown that Dox-PSA loading in LTSL can block its biological activity at 37°C, while the combination with mild HT resulted in augmented cytotoxicity in both 2D and 3D PC models compared to the free Dox-PSA. More importantly, Dox-PSA encapsulation in LTSL prolonged its blood circulation and reduced Dox accumulation in the heart of C4-2B tumor-bearing mice over the free Dox-PSA, thus significantly improving Dox-PSA therapeutic window. Finally, Dox-PSA-loaded LTSL combined with HT significantly delayed tumor growth at a similar rate as mice treated with free Dox-PSA in both solid and metastatic PC tumor models. This indicates this strategy could block the systemic cleavage of Dox-PSA without reducing its efficacy in vivo, which could represent a safer option to treat patients with locally advanced PC. STATEMENT OF SIGNIFICANCE: This study investigates a new tactic to tackle non-specific cleavage of doxorubicin PSA-activatable prodrug (L-377,202) to treat advanced prostate cancer. In the present study, we report a nanoparticle-based approach to overcome the non-specific activation of L-377,202 in the systemic circulation. This includes encapsulating Dox-PSA in low temperature-sensitive liposomes to prevent its premature hydrolysis and non-specific cleavage. This class of liposomes offers payload protection against degradation in plasma, improved pharmacokinetics and tumor targeting, and an efficient and controlled drug release triggered by mild hyperthermia (HT) (∼42°C). We believe that this strategy holds great promise in bypassing any systemic toxicity concerns that could arise from the premature activation of the prodrug whilst simultaneously being able to control the spatiotemporal context of Dox-PSA cleavage and metabolism.

Nanoprecipitation preparation of low temperature-sensitive magnetoliposomes.

Lysolipid-containing thermosensitive liposomes (LTSL) have gained attention for triggered release of chemotherapeutics. Superparamagnetic iron oxide nanoparticles (SPION) offers multimodal imaging and hyperthermia therapy opportunities as a promising theranostic agent. Combining LTSL with SPION may further enhance their performance and functionality of LTSL. However, a major challenge in clinical translation of nanomedicine is the poor scalability and complexity of their preparation process. Exploiting the nature of self-assembly, nanoprecipitation is a simple and scalable technique for preparing liposomes. Herein, we developed a novel SPION-incorporated lysolipid-containing thermosensitive liposome (mLTSL10) formulation using nanoprecipitation. The formulation and processing parameters were carefully designed to ensure high reproducibility and stability of mLTSL10. The effect of solvent, aqueous-to-organic volume ratio, SPION concentration on the mLTSL10 size and dispersity was investigated. mLTSL10 were successfully prepared with a small size (∼100 nm), phase transition temperature at around 42 °C, and high doxorubicin encapsulation efficiency. Indifferent from blank LTSL, we demonstrated that mLTSL10 combining the functionality of both LTSL and SPION can be successfully prepared using a scalable nanoprecipitation approach.

Hypoxia-targeted cupric-tirapazamine liposomes potentiate radiotherapy in prostate cancer spheroids.

In this study, novel cupric-tirapazamine [Cu(TPZ)2]-liposomes were developed as an effective hypoxia-targeted therapeutic, which potentiated radiotherapy in a three dimensional (3D) prostate cancer (PCa) model. To overcome the low water solubility of the Cu(TPZ)2, a remote loading method was developed to efficiently load the lipophilic complex into different liposomal formulations. The effect of pH, temperature, PEGylation, lipid composition, liposome size, lipid: complex ratio on the liposome properties, and drug loading was evaluated. The highest loading efficiency was obtained at neutral pH, which was independent of lipid composition and incubation time. In addition, enhanced drug loading was achieved upon decreasing the lipid:complex molar ratio with minimal effects on liposomes' morphology. Interestingly, the in vitro potency of the developed liposomes was easily manipulated by changing the lipid composition. The hydrophilic nature of our liposomal formulations improved the complex's solubility, leading to enhanced cellular uptake and toxicity, both in PCa monolayers and tumour spheroids. Moreover, Cu(TPZ)2-loaded liposomes combined with radiation, showed a significant reduction in PCa spheroids growth rate, compared to the free complex or radiation alone, which could potentiate radiotherapy in patients with localised advanced PCa.

PD1 blockade potentiates the therapeutic efficacy of photothermally-activated and MRI-guided low temperature-sensitive magnetoliposomes.

This study investigates the effect of PD1 blockade on the therapeutic efficacy of novel doxorubicin-loaded temperature-sensitive liposomes. Herein, we report photothermally-activated, low temperature-sensitive magnetoliposomes (mLTSL) for efficient drug delivery and magnetic resonance imaging (MRI). The mLTSL were prepared by embedding small nitrodopamine palmitate (NDPM)-coated iron oxide nanoparticles (IO NPs) in the lipid bilayer of low temperature-sensitive liposomes (LTSL), using lipid film hydration and extrusion. Doxorubicin (DOX)-loaded mLTSL were characterized using dynamic light scattering, differential scanning calorimetry, electron microscopy, spectrofluorimetry, and atomic absorption spectroscopy. Photothermal experiments using 808 nm laser irradiation were conducted. In vitro photothermal DOX release studies and cytotoxicity was assessed using flow cytometry and resazurin viability assay, respectively. In vivo DOX release and tumor accumulation of mLTSL(DOX) were assessed using fluorescence and MR imaging, respectively. Finally, the therapeutic efficacy of PD1 blockade in combination with photothermally-activated mLTSL(DOX) in CT26-tumor model was evaluated by monitoring tumor growth, cytokine release and immune cell infiltration in the tumor tissue. Interestingly, efficient photothermal heating was obtained by varying the IO NPs content and the laser power, where on-demand burst DOX release was achievable in vitro and in vivo. Moreover, our mLTSL exhibited promising MR imaging properties with high transverse r2 relaxivity (333 mM-1 s-1), resulting in superior MR imaging in vivo. Furthermore, mLTSL(DOX) therapeutic efficacy was potentiated in combination with anti-PD1 mAb, resulting in a significant reduction in CT26 tumor growth via immune cell activation. Our study highlights the potential of combining PD1 blockade with mLTSL(DOX), where the latter could facilitate chemo/photothermal therapy and MRI-guided drug delivery.

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes.

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (> 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 °C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

Organ Biodistribution of Radiolabelled γδ T Cells Following Liposomal Alendronate Administration in Different Mouse Tumour Models.

Vγ9Vδ2 T cell immunotherapy has been shown to be effective in delaying tumour growth in both pre-clinical and clinical studies. It has been pointed out the importance of the ability of cells to accumulate within tumours and the association with therapeutic efficacy in clinical studies of adoptive T cell transfer. We have previously reported that alendronate liposomes (L-ALD) increase the efficacy of this therapy after localised or systemic injection of γδ T cells in mice, inoculated with ovarian, melanoma, pancreatic or experimental lung metastasis tumour models, respectively. This study aimed to examine the organ biodistribution and tumour uptake of human γδ T cells in subcutaneous (SC), intraperitoneal (IP) or experimental metastatic lung tumours, established in NOD-SCID gamma (NSG) mice using the melanoma cell line A375Pß6.luc. pre-injected with L-ALD. Overall, small variations in blood profiles and organ biodistribution of γδ T cells among the different tumour models were observed. Exceptionally, IP-tumour and experimental metastatic lung-tumour bearing mice pre-injected with L-ALD showed a significant decrease in liver accumulation, and highest uptake of γδ T cells in lungs and tumour-bearing lungs, respectively. Lower γδ T cell count was found in the SC and IP tumours.

Encapsulated doxorubicin crystals influence lysolipid temperature-sensitive liposomes release and therapeutic efficacy in vitro and in vivo.

Doxorubicin (DOX)-loaded lysolipid temperature-sensitive liposomes (LTSLs) are a promising stimuli-responsive drug delivery system that rapidly releases DOX in response to mild hyperthermia (HT). This study investigates the influence of loaded DOX crystals on the thermosensitivity of LTSLs and their therapeutic efficacy in vitro and in vivo. The properties of DOX crystals were manipulated using different remote loading methods (namely (NH4)2SO4, NH4-EDTA and MnSO4) and varying the lipid:DOX weight ratio during the loading step. Our results demonstrated that (NH4)2SO4 or NH4-EDTA remote loading methods had a comparable encapsulation efficiency (EE%) into LTSLs in contrast to the low DOX EE% obtained using the metal complexation method. Cryogenic transmission electron microscopy (cryo-TEM) revealed key differences in the nature of DOX crystals formed inside LTSLs based on the loading buffer or/and the lipid:DOX ratio used, resulting in different DOX release profiles in response to mild HT. The in vitro assessment of DOX release/uptake in CT26 and PC-3 cells revealed that the use of a high lipid:DOX ratio exhibited a fast and controlled release profile in combination with mild HT, which correlated well with their cytotoxicity studies. Similarly, in vivo DOX release, tumour growth inhibition and mice survival rates were influenced by the physicochemical properties of LTSLs payload. This study demonstrates, for the first time, that the characteristics of DOX crystals loaded into LTSLs, and their conformational rearrangement during HT, are important factors that impact the TSLs performance in vivo.

Liposome-Templated Indocyanine Green J- Aggregates for In Vivo Near-Infrared Imaging and Stable Photothermal Heating.

Indocyanine green (ICG) is an FDA-approved near-infrared fluorescent dye that has been used in optical imaging and photothermal therapy. Its rapid in vivo clearance and photo-degradation have limited its application. ICG pharmacokinetics and biodistribution have been improved via liposomal encapsulation, while its photothermal stability has been enhanced by ICG J-aggregate (IJA) formation. In the present work, we report a simple approach to engineer a nano-sized, highly stable IJA liposomal formulation. Our results showed that lipid film hydration and extrusion method led to efficient IJA formation in rigid DSPC liposomes, as supported by molecular dynamics modeling. The engineered DSPC-IJA formulation was nano-sized, and with spectroscopic and photothermal properties comparable to free IJA. Promisingly, DSPC-IJA exhibited high fluorescence, which enabled its in vivo tracking, showing prolonged blood circulation and significantly higher tumor fluorescence signals, compared to free ICG and IJA. Furthermore, DSPC-IJA demonstrated high photo-stability in vivo after multiple cycles of 808 nm laser irradiation. Finally, doxorubicin was loaded into liposomal IJA to utilize the co-delivery capabilities of liposomes. In conclusion, with both liposomes and ICG being clinically approved, our novel liposomal IJA could offer a clinically relevant theranostic platform enabling multimodal imaging and combinatory chemo- and photothermal cancer therapy.

EGFR-targeted immunoliposomes efficiently deliver docetaxel to prostate cancer cells.

Prostate cancer is the second cause of cancer death in men worldwide. Docetaxel (DTX), an antimitotic drug, is widely used for the treatment of metastatic prostate cancer patients. Taxotere® is a commercial DTX formulation. It contains a polysorbate 80 surfactant to improve DTX aqueous solubility, which has been associated with hypersensitivity reactions in patients. Liposomes have been used as promising delivery systems for a range of hydrophobic drugs, such as DTX, offering improved drug water solubility and biocompatibility, without compromising its anticancer activity. Herein, DTX-loaded liposomes were developed using the Box-Behnken factorial design. The optimized formulation was nano-sized, homogenous in size (67.47 nm) with high DTX encapsulation efficiency (99.95 %). The encapsulated DTX was in a soluble amorphous state, which was slowly released. Next, to increase the liposomes selectivity to prostate cancer cells, cetuximab, an anti-EGFR monoclonal antibody. was successfully conjugated to the surface of liposomes, without compromising cetuximab protein structure and stability. As expected, our results showed higher cellular uptake and toxicity of immunoliposomes, compared to non-targeted liposomes, in DU145 (EGFR-overxpressing) prostate cancer cells. To the best of our knowledge, this is the first report of engineering EGFR-targeted liposomes to enhance the selectivity of DTX delivery to EGFR-positive prostate cancer cells.

Blood circulation and tissue biodistribution of lipid--quantum dot (L-QD) hybrid vesicles intravenously administered in mice.

The present work describes the pharmacokinetics of recently developed liposome-quantum dot (L-QD) hybrid vesicles in nude mice following systemic administration. Hydrophobic QD were incorporated into different bilayer compositions, and the serum stability of such hybrid vesicles was evaluated using turbidity and carboxyfluorescein release measurements. L-QD hybrid blood profile and organ biodistribution were also determined by elemental (cadmium) analysis. Following intravenous administration, different tissue biodistribution profiles and tissue affinities were observed depending on the L-QD lipid bilayer characteristics. Immediate blood clearance was observed with cationic (DOTAP/DOPE/Chol) hybrid with rapid lung accumulation, while incorporation of PEG at the surface of zwitterionic vesicles dramatically prolonged their blood circulation half-life after systemic administration. Overall, the L-QD hybrid vesicle system is considered a viable platform that allows QD delivery to different tissues through facile modulation of the hybrid vesicle characteristics. In addition, L-QD offers many opportunities for the development of combinatory therapeutic and imaging (theranostic) modalities by incorporating both drug molecules and QD within the different compartments of a single vesicle.

Sterically stabilized liposomes production using staggered herringbone micromixer: Effect of lipid composition and PEG-lipid content.

Preparation of lipid-based drug delivery systems by microfluidics has been increasingly popular, due to the reproducible, continuous and scalable nature of the microfluidic process. Despite exciting development in the field, versatility and superiority of microfluidics over conventional methods still need further evidence, since preparing clinically-relevant sterically stabilised liposomes has been lacking. The present study describes the optimisation of PEGylated liposomal formulations of various rigidity using staggered herringbone micromixer (SHM). The effect of both processing parameters (total flow rate (TFR) and aqueous-to-ethanol flow rate ratio (FRR)) and formulation parameters (lipid components and composition, initial lipid concentration and aqueous media) was investigated and discussed. Liposomal formulations consist of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), with cholesterol and PEGylated lipid (DSPE-PEG2000) were successfully prepared with the desired size (∼100 nm) and dispersity (<0.2). Doxorubicin was successfully encapsulated in these liposomes at high (>80%) encapsulation efficiency using the pH-gradient remote loading method, illustrating their bilayer integrity and capability as drug delivery systems. We demonstrated that clinically-relevant PEGylated liposomal formulations could be prepared with properties comparable to conventional techniques. Limitations and recommendations on the microfluidic production of PEGylated liposomes were also discussed.

Functionalized-quantum-dot-liposome hybrids as multimodal nanoparticles for cancer.

Functionalized-quantum-dot-liposome (f-QD-L) hybrid nanoparticles are engineered by encapsulating poly(ethylene glycol)-coated QD in the internal aqueous phase of different lipid bilayer vesicles. f-QD-L maintain the QD fluorescence characteristics as confirmed by fluorescence spectroscopy, agarose gel electrophoresis, and confocal laser scanning microscopy. Cationic f-QD-L hybrids lead to dramatic improvements in cellular binding and internalization in tumor-cell monolayer cultures. Deeper penetration into three-dimensional multicellular spheroids is obtained for f-QD-L by modifying the lipid bilayer characteristics of the hybrid system. f-QD-L are injected intratumorally into solid tumor models leading to extensive fluorescent staining of tumor cells compared to injections of the f-QD alone. f-QD-L hybrid nanoparticles constitute a versatile tool for very efficient labeling of cells ex vivo and in vivo, particularly when long-term imaging and tracking of cells is sought. Moreover, f-QD-L offer many opportunities for the development of combinatory therapeutic and imaging (theranostic) modalities by incorporating both drug molecules and QD within the different compartments of a single vesicle.

Construction of nanoscale multicompartment liposomes for combinatory drug delivery.

Liposomes are clinically used delivery systems for chemotherapeutic agents, biological macromolecules and diagnostics. Due to their flexibility in size and composition, different types of liposomes have been developed varying in surface and structural characteristics. Multicompartment liposomes constitute an attractive drug carrier system offering advantages in terms of inner vesicle protection, sustained drug release and possibility for combinatory (cocktail) therapies using a single delivery system. However, all previously described methodologies for multicompartment or multivesicular liposomes resulted in micrometer-sized vesicles limiting most pharmaceutical applications. In this work we report formulation of nanoscale multicompartment liposomes which maybe applicable for systemic administration. A small unilamellar vesicle (SUV) aqueous dispersion (DOPC:DOPG:CHOL) was used to hydrate a dried film of different lipid contents (DMPC:CHOL), followed by extrusion. The system was characterised by techniques such as photon correlation spectroscopy (PCS), zeta potential measurement, transmission electron microscopy (TEM) and laser scanning confocal microscopy (LSCM). We observed a single, multicompartment vesicle population composed of the two different bilayer types of approximately 200 nm in mean diameter rather than a mixture of two independent vesicle populations. In the case of tumour therapy, such multicompartment liposome systems can offer a single carrier for the delivery of two different modalities.

Investigating in vitro and in vivo αvß6 integrin receptor-targeting liposomal alendronate for combinatory γδ T cell immunotherapy.

The αvß6 integrin receptor has been shown to be overexpressed on many types of cancer cells, resulting in a more pro-invasive and aggressive phenotype, this makes it an attractive target for selective drug delivery. In tumours that over-express the αvß6 receptor, cellular uptake of liposomes can be enhanced using ligand-targeted liposomes. It has previously been shown in both in vitro and in vivo studies that liposomal alendronate (L-ALD) can sensitise cancer cells to destruction by Vγ9Vδ2 T cells. It is hypothesised that by using the αvß6-specific peptide A20FMDV2 as a targeting moiety for L-ALD, the therapeutic efficacy of this therapy can be increased in αvß6 positive tumours. Targeted liposomes (t-L) were formulated and the targeting efficacy of targeted liposomes (t-L) was assessed by cell uptake and cytotoxicity studies in the αvß6 positive cells line A375Pß6. Bio-distribution of both L and t-L were carried out in αvß6 positive (A375Pß6 and PANC0403) and αvß6 negative (A375Ppuro and PANC-1) subcutaneous tumour mouse models. Immuno-compromised mice bearing A375Pß6 experimental metastatic lung tumours were treated with L-ALD or t-L-ALD as monotherapies or in combination with ex vivo-expanded Vγ9Vδ2 T cells. In vitro, αvß6-dependant uptake of t-L was observed, with t-L-ALD being more effective than L-ALD at sensitising A375Pß6 to γδ T cells. Interestingly, t-L-ALD led to slightly higher but not significant reduction in tumour growth compared to L-ALD, when used as monotherapy in vivo. Moreover, both L-ALD and t-L-ALD led to significant reductions in tumour growth when used in combination with γδ T cells in vivo but t-L-ALD offered no added advantage compared to L-ALD.