Dually targeted bioinspired nanovesicle delays advanced prostate cancer tumour growth in vivo.

Prostate cancer (PC) is second-leading cancer in men, with limited treatment options available for men with advanced and metastatic PC. Prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) have been exploited as therapeutic targets in PC due to their upregulation in the advanced stages of the disease. To date, several PSA- and PSMA-activatable prodrugs have been developed to reduce the systemic toxicity of existing chemotherapeutics. Bioinspired nanovesicles have been exploited in drug delivery, offering prolonged drug blood circulation and higher tumour accumulation. For the first time, this study describes the engineering of dually targeted PSA/PSMA nanovesicles for advanced PC. PSMA-targeted bioinspired hybrids were prepared by hydrating a lipid film with anti-PSMA-U937 cell membranes and DOX-PSA prodrug, followed by extrusion. The bioinspired hybrids were characterised using dynamic light scattering, transmission electron microscopy, Dot blot, flow cytometry and Western blot. Cellular binding and toxicity studies in PC cancer cell lines were carried out using flow cytometry, confocal microscopy, and resazurin assay. Finally, tumour targeting and therapeutic efficacy studies were performed in solid and metastatic C4-2B-tumor-bearing mice. Interestingly, our PSMA-targeted hybrids demonstrated high cell uptake in PSMA-expressing cells with significant accumulation in solid and metastatic C4-2B tumour tissues following intravenous administration. More promisingly, our dually targeted PSA/PSMA hybrid significantly slowed down the C4-2B tumour growth in vivo, compared to free DOX-PSA and non-targeted PSA-hybrid. Our PSA/PSMA bioinspired hybrid could offer a highly selective treatment for advanced PC with lower side effects. STATEMENT OF SIGNIFICANCE: This study investigates a new approach to treat prostate cancer using dually targeted bioinspired nanovesicle . Our bioinspired vesicles are made mainly of a human blood cell membrane with a ligand recognising a specific marker (PSMA) on the surface of the prostate cancer cells. The present work describes the successful loading of a doxorubicin prodrug linked to a PSA- activatable peptide into these targeted bioinspired nanovesicle , where the active PSA enzyme presents in these cells converts the drug to its active form. Our dually targeted PSA/PSMA hybrid vesicles has successfully improved site-specific prodrug delivery to tackle advanced prostate cancer, offering a novel and effective prostate cancer treatment.

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.

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.

Genetically-engineered anti-PSMA exosome mimetics targeting advanced prostate cancer in vitro and in vivo.

The present work describes the engineering of anti-PSMA peptide-decorated exosome mimetics (EMs) targeting advanced prostate cancer (PC). The targeted EMs were produced from anti-PSMA peptide, WQPDTAHHWATL, expressing U937 monoblastic cells, followed by successive extrusion cycles. The engineered EMs were nanosized, produced at a high yield, and displayed the anti-PSMA peptide, exosomal markers and monocytes proteins on their surface. As anticipated, PSMA-EMs showed increased cellular internalization in PSMA positive PC cell lines (LNCaP and C4-2B), compared to unmodified EMs. Most importantly, higher tumour targeting was observed in solid C4-2B tumours, following intravenous administration, confirming their targeting ability in vivo. Overall, our study indicates that the engineered anti-PSMA peptide-targeted EMs can be a promising drug delivery system for advanced PC.

Magneto-Liposomes as MRI Contrast Agents: A Systematic Study of Different Liposomal Formulations.

The majority of the clinically approved iron oxide nanoparticles (IO NPs) used as contrast agents for magnetic resonance imaging (MRI) have been withdrawn from the market either due to safety concerns or lack of profits. To address this challenge, liposomes have been used to prepare IO-based T2 contrast agents. We studied the influence of different phospholipids on the relaxivity (r2) values of magneto-liposomes (MLs) containing magnetic NPs in the bilayer, where a strong correlation between the bilayer fluidity and r2 is clearly shown. Embedding 5-nm IO NPs in the lipid bilayer leads to a significant improvement in their relaxivity, where r2 values range from 153 ± 5 s-1 mM-1 for DPPC/cholesterol/DSPE-PEG (96/50/4) up to 673 ± 12 s-1 mM-1 for DOPC/DSPE-PEG (96/4), compared to "free" IO NPs with an r2 value of 16 s-1 mM-1, measured at 9.4 T MRI scanner. In vitro MRI measurements, together with the ICP-MS analysis, revealed MLs as highly selective contrast agents that were preferentially taken up by cancerous T24 cells, which led to an improvement in the contrast and an easier distinction between the healthy and the cancerous cells. A careful selection of the lipid bilayer to prepare MLs could offer efficient MRI contrast agents, even at very low IO NP concentrations.

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.

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.

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.