Nano-modified viruses prime the tumor microenvironment and promote the photodynamic virotherapy in liver cancer.

BACKGROUND: As of 2020, hepatocellular carcinoma (HCC), a form of liver cancer, stood as the third most prominent contributor to global cancer-related mortality. Combining immune checkpoint inhibitors (ICI) with other therapies has shown promising results for treating unresectable HCC, offering new opportunities. Recombinant adeno-associated viral type 2 (AAV2) virotherapy has been approved for clinical use but it efficacy is stifled through systemic administration. On the other hand, iron oxide nanoparticles (ION) can be cleared via the liver and enhance macrophage polarization, promoting infiltration of CD8+ T cells and creating a more favorable tumor microenvironment for immunotherapy. METHODS: To enhance the efficacy of virotherapy and promote macrophage polarization towards the M1-type in the liver, ION-AAV2 were prepared through the coupling of ION-carboxyl and AAV2-amine using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/N-hydroxysulfosuccinimide (Sulfo-NHS). Efficacy after systemic delivery of ION-AAV2 in an orthotopic HCC model was evaluated. RESULTS: After 28 days, the tumor weight in mice treated with ION-AAV2 was significantly reduced by 0.56-fold compared to the control group. The ION-AAV2 treatment led to an approximate 1.80-fold increase in the level of tumor associated M1-type macrophages, while the number of M2-type macrophages was reduced by 0.88-fold. Moreover, a proinflammatory response increased the population of tumor-infiltrating CD8+ T cells in the ION-AAV2 group. This transformation converted cold tumors into hot tumors. CONCLUSIONS: Our findings suggest that the conjugation of ION with AAV2 could be utilized in virotherapy while simultaneously exploiting macrophage-modulating cancer immunotherapies to effectively suppress HCC growth.

Potential targeting of the tumor microenvironment to improve cancer virotherapy.

In 2015, oncolytic virotherapy was approved for clinical use, and in 2017, recombinant adeno-associated virus (AAV) delivery was also approved. However, systemic administration remains challenging due to the limited number of viruses that successfully reach the target site. Although the US Food and Drug Administration (FDA) permits the use of higher doses of AAV to achieve greater rates of transduction, most AAV still accumulates in the liver, potentially leading to toxicity there and elsewhere. Targeting the tumor microenvironment is a promising strategy for cancer treatment due to the critical role of the tumor microenvironment in controlling tumor progression and influencing the response to therapies. Newly discovered evidence indicates that administration routes focusing on the tumor microenvironment can promote delivery specificity and transduction efficacy within the tumor. Here, we review approaches that involve modifying viral surface features, modulating the immune system, and targeting the physicochemical characteristics in tumor microenvironment to regulate therapeutic delivery. Targeting tumor acidosis presents advantages that can be leveraged to enhance virotherapy outcomes and to develop new therapeutic approaches that can be integrated with standard treatments.

Comparing the Variants of Iron Oxide Nanoparticle-Mediated Delivery of miRNA34a for Efficiency in Silencing of PD-L1 Genes in Cancer Cells.

The blocking of programmed death-ligand 1 (PD-L1) in tumor cells represents a powerful strategy in cancer immunotherapy. Using viral vectors to deliver the cargo for inactivating the PD-L1 gene could be associated with host cell genotoxicity and concomitant immune attack. To develop an alternative safe gene delivery method, we designed a unique combination for miRNA34a delivery using a transgene carrier in the form of iron oxide magnetic nanoparticles (IONPs) via magnetofection to downregulate PD-L1 expression in cancer cells. We synthesized IONPs of multiple shapes (IONRs (iron oxide nanorods), IONSs (iron oxide nanospheres), and ITOHs (iron oxide truncated octahedrons)), surface-functionalized with polyethyleneimine (PEI) using the ligand exchange method, as gene delivery systems. Under the guidance of an external magnetic field, PEI@IONPs loaded with plasmid DNA (DNA/PEI@IONPs) encoding GFP showed high transfection efficiency at different weight ratios and time points in A549 and MDA-MB-231 cells. Additionally, the DNA/PEI@IONPs with miRNA34a inserts under a static magnetic field resulted in significant knockdown of the PD-L1 gene, as demonstrated via immunoblotting of the PD-L1 protein. Among the three shapes of IONPs, IONRs showed the highest PD-L1 knockdown efficiency. The genetic expression of miRNA34a was also studied using qPCR and it showed high expression of miRNA in cells treated with PEI@IONRs. Flow cytometry and a live/dead assay confirmed apoptosis after transfection with miRNA34a. To conclude, in this paper, a promising transgene carrier with low cost, negligible cytotoxicity, and high transfection efficiency has been successfully established for miRNA gene delivery in the context of cancer immunotherapy.

Light- and Redox-Responsive Block Copolymers of mPEG-SS-ONBMA as a Smart Drug Delivery Carrier for Cancer Therapy.

The development of stimuli-responsive polymeric micelles for targeted drug delivery has attracted much research interest in improving therapeutic outcomes. This study designs copolymers responsive to ultraviolet (UV) light and glutathione (GSH). A disulfide linkage is positioned between a hydrophilic poly(ethylene glycol) monomethyl ether (mPEG) and a hydrophobic o-nitrobenzyl methacrylate (ONBMA) to yield amphiphilic copolymers termed mPEG-SS-pONBMA. Three copolymers with different ONBMA lengths are synthesized and formulated into micelles. An increase in particle size and a decrease in critical micelle concentration go together with increasing ONBMA lengths. The ONB cleavage from mPEG-SS-pONBMA-formed micelles results in the transformation of hydrophobic cores into hydrophilic ones, accelerating drug release from the micelles. Obvious changes in morphology and molecular weight of micelles upon combinational treatments account for the dual-stimuli responsive property. Enhancement of a cell-killing effect is clearly observed in doxorubicin (DOX)-loaded micelles containing disulfide bonds compared with those containing dicarbon bonds upon UV light irradiation. Collectedly, the dual-stimuli-responsive mPEG-SS-pONBMA micelle is a better drug delivery carrier than the single-stimuli-responsive mPEG-CC-pONBMA micelle. After HT1080 cells were treated with the DOX-loaded micelles, the high expression levels of RIP-1 and MLKL indicate that the mechanism involved in cell death is mainly via the DOX-induced necroptosis pathway.

Macrophage Distribution Affected by Virus-Encoded Granulocyte Macrophage Colony Stimulating Factor Combined with Lactate Oxidase.

Oncolytic virotherapy was approved as a localized treatment for advanced melanoma by the US Food and Drug Administration (FDA) in 2015. Granulocyte macrophage colony stimulating factor (GM-CSF) encoded by clinical virus-infected tumor cells, acting as a pro-inflammatory cytokine or growth factor, increases tumor antigen presentation, leading to the activation of macrophages and T cells. Notably, tumor-secreted lactate can promote the suppressive functions of M2-polarized tumor-associated macrophages and subsequently promote tumor growth. Furthermore, the consumption of tumor-secreted lactate has been implicated in the beneficial polarization of macrophages. Here, we report that GM-CSF-encoded recombinant adeno-associated virus (AAV2-GM-CSF) infection in B16-F10 mouse melanoma cells combined with lactate oxidase (LOX) leads to the recruitment of M1 macrophages for the inhibition of cancer cell growth. This study suggests that GM-CSF combined with LOX has potential as cancer virotherapy.

Peripheral benzodiazepine receptor TSPO needs to be reconsidered before using as a drug target for a pigmentary disorder.

The peripheral benzodiazepine receptor (TSPO/PBR) is highly conserved among different species but with perplexing biochemical functions. Multiple ligands of TSPO show commendable regulatory activities in lots of biological functions, such as neuro-protection, cholesterol transport, and so on. These researches support that TSPO may be a potential target for disease treatment and drug development. Previous studies have shown that its ligands benzodiazepines show a satisfactory effect on melanogenic promotion. However, the potential application of TSPO in drug development for pigmentary disorder needs further investigation. In this study, we confirmed the melanogenesis induction of TSPO ligand, Ro5-4864 in mouse melanoma cell lines, human skin tissue, and zebrafish embryos by inducing melanin synthesis and melanosome transport. Molecular genetics and pharmacological studies showed that TSPO deficiency did not affect melanin production in B16F10 cells and zebrafish embryos, nor did it affect the melanin promotion effect of Ro5-4864. Whether or not TSPO exists, the expression of lots of melanogenesis-related proteins, such as TYR, TRP-1, DCT, Mlph, and Rab27 was upregulated with the Ro5-4864 administration. These results indicated that Ro5-4864 induces melanogenesis in a TSPO-independent manner, which is inconsistent with previous research. This research is a reminder that we need to be very careful during target validation in drug development.

Synergistic Effect of Repolarization of M2 to M1 Macrophages Induced by Iron Oxide Nanoparticles Combined with Lactate Oxidase.

Metabolic reprogramming of tumors with the accompanying reprogramming of glucose metabolism and production of lactate accumulation is required for the subsequent development of tumors. Recent evidence has indicated that tumor-secreted lactate can promote an oncolytic immune microenvironment within the tumor. Furthermore, tumor-secreted lactate directly induces polarization of tumor-supportive M2 macrophages. However, oxidized tumor-secreted lactate in the tumor microenvironment can be exploited. Iron oxide nanoparticles have shown promising anticancer potential by activating tumor-suppressing macrophages. Furthermore, lactate oxidase (LOX) generally oxidizes tumor-secreted lactate and subsequently converts to pyruvate. Particularly, the ratio of M2 macrophages to M1 macrophages corresponds with tumor growth. In this study, we present iron oxide nanoparticles with carboxylic acid combined with LOX that enhance antitumor efficacy as a synergistic effect on the repolarization of tumor-supportive M2 macrophages to tumor-suppressive M1 macrophages in a tumor microenvironment. After M2 macrophages treated with iron oxide nanoparticles were combined with LOX, the ratio of M1 macrophages was significantly greater than iron oxide nanoparticles alone or with LOX alone. It is concluded that the inhibition of cancer cell proliferation by ratio of M1 macrophages was observed. This study suggests that the iron oxide nanoparticles combined with LOX could be potentially used for potentiating immune checkpoint inhibitor therapies for cancer treatment.

Potential therapeutics using tumor-secreted lactate in nonsmall cell lung cancer.

Targeted-therapy failure in treating nonsmall cell lung cancer (NSCLC) frequently occurs because of the emergence of drug resistance and genetic mutations. The same mutations also result in aerobic glycolysis, which further antagonizes outcomes by localized increases in lactate, an immune suppressor. Recent evidence indicates that enzymatic lowering of lactate can promote an oncolytic immune microenvironment within the tumour. Here, we review factors relating to lactate expression in NSCLC and the utility of lactate oxidase (LOX) for governing therapeutic delivery, its role in lactate oxidation and turnover, and relationships between lactate depletion and immune cell populations. The lactate-rich characteristic of NSCLC provides an exploitable property to potentially improve NSCLC outcomes and design new therapeutic strategies to integrate with conventional therapies.

ROP and ATRP fabricated redox sensitive micelles based on PCL-SS-PMAA diblock copolymers to co-deliver PTX and CDDP for lung cancer therapy.

Combining dual drugs in one vehicle to cancer cells offers spatiotemporal localization of drug at the site of action, leading to synergistic therapeutic effects and reduced side effects. To improve pH/redox responsiveness to the tumor microenvironments for cancer therapy, a pH/redox-responsive micelle based on poly(ε-caprolactone)-SS-poly(methacrylic acid) (PCL-SS-PMAA) diblock copolymer was fabricated for dual drug delivery. The PCL-SS-PMAA was formulated into a core-shell micelle (PSPm) in an aqueous solution. The critical micelle concentration (CMC) values of PSPm were 7.94 × 10-3 mg mL-1 at pH 5.0 and 1.00 × 10-2 mg mL-1 at pH 7.4. The hydrodynamic diameters of PSPm were within 210-270 nm, depending on pH values. Changes in morphology and size of PSPm were clearly observed before and after exposure to a reducing agent. Paclitaxel (PTX) was encapsulated into the core and cisplatin (CDDP) was chelated on the shell of PSPm, with both PTX and CDDP being efficiently released from PSPm in the presence of a reducing agent in an acid condition. MTT and annexin V/propidium iodide dual staining results demonstrated that co-loading of CDDP and PTX into PSPm had a synergistic effect in killing lung cancer cells and exerted superior antitumor activity over the combination of single drug-loaded PSPm or the combination of free-CDDP and free-PTX at equivalent drug amounts. Hence, encapsulating the dual drugs into PSPm exhibits a synergistic effect for potential lung cancer therapy.

Dual Stimuli-Responsive Block Copolymers with Adjacent Redox- and Photo-Cleavable Linkages for Smart Drug Delivery.

A novel dual-stimuli cleavable linker containing adjacent UV light-sensitive o-nitrobenzyl ester and GSH-responsive disulfide bonds was first designed and synthesized to increase the responsivity to external stimuli. The functionalized linker was then utilized to prepare a dual-responsive amphiphilic block copolymer using ring-opening polymerization and atom transfer radical polymerization. The copolymer formed a micelle in an aqueous solution and showed dual-stimuli responses including photo-mediated cleavage under UV light irradiation at 365 nm as well as reduction-responsive degradation in the presence of a reducing agent. The micelle was nontoxic against three cell lines and majorly internalized via clathrin-mediated endocytosis. Doxorubicin (Dox) was loaded in the hydrophobic core of the micelle. Enhancement of a cell-killing effect against A549 cells was clearly observed in the Dox-encapsulated micelle when exposed to UV light.

Nanomodified strategies to overcome EGFR-tyrosine kinase inhibitors resistance in non-small cell lung cancer.

Lung cancer is the primary cause of cancer-related death worldwide. 85%-90% of cases are non-small cell lung cancer (NSCLC) which characteristically exhibits altered epidermal growth factor receptor (EGFR) signaling is a major driver pathway. Unfortunately, therapeutic outcomes in treating NSCLC are compromised by the emergence of drug resistance in response to EGFR-tyrosine kinase inhibitor (TKI) targeted therapy due to the acquired resistance mutation EGFR T790M or activation of alternative pathways. There is current need for a new generation of TKIs to be developed to treat EGFR-TKI-resistant NSCLC. To overcome the above problems and improve clinical efficacy, nanotechnology with targeting abilities and sustained release has been proposed for EGFR-TKI-resistant NSCLC treatment and has already achieved success in in vitro or in vivo models. In this review, we summarize and illustrate representative nano-formulations targeting EGFR-TKI-resistant NSCLC. The described advances may pave the way to better understanding and design of nanocarriers and multifunctional nanosystems for efficient treatment for drug resistant NSCLC.

Repolarization of M2 to M1 Macrophages Triggered by Lactate Oxidase Released from Methylcellulose Hydrogel.

Deregulated proliferation of tumors is generally associated with altered energy metabolism. A high rate of anaerobic glycolysis in solid tumors contributes to an acidification of pH to ∼6.7-7.2 in the tumor microenvironment and lactate accumulation. Macrophages in the tumor microenvironment can be educated by tumor cells. Tumor-derived lactate induces the polarization of M2 macrophages and promotes tumor invasion and metastasis. However, a particular challenge is to sustain lactate depletion. We propose that the repolarization of the tumor-supportive M2 macrophage to the tumor-suppressive M1 macrophage after the depletion of lactate by lactate oxidase (LOX) released from the hydrogels in the tumor microenvironment may enhance the antitumor treatment efficacy.

CS-PEI/Beclin-siRNA Downregulate Multidrug Resistance Proteins and Increase Paclitaxel Therapeutic Efficacy against NSCLC.

Paclitaxel (PTX) is a widely used chemotherapy drug; however, frequent use causes multidrug resistance (MDR), which limits the utility of PTX against advanced non-small-cell lung cancer (NSCLC). PTX-resistant subline (NCI-H23-TXR) was established in vitro by exposing NCI-H23 cells to gradually increased concentrations of PTX in culture medium. Distinct Beclin expression of autophagy level was observed between resistant NCI-H23-TXR and parental NCI-H23 cells. Beclin-small interfering RNA (siRNA) was selected to restore sensitivity of PTX against NCI-H23-TXR. Chondroitin sulfate-polyethylenimine (CS-PEI) was constructed for delivery and protection of Beclin-siRNA. To delineate the underlying molecular mechanism of Beclin knockdown, we analyzed different MDR expression proteins of two cells using western blot, and the corresponding genes were confirmed by real-time PCR. Compared with NCI-H23, NCI-H23-TXR had higher expression levels in P-glycoprotein (P-gp) and multidrug resistance protein 7 (ABCC10). Knockdown of Beclin simultaneously inhibited P-gp and ABCC10, and renewed the sensitivity of PTX against NCI-H23-TXR. Research on zebrafish embryos revealed that tumor sizes decreased in NCI-H23 tumor xenografts but remained intact in NCI-H23-TXR tumor xenografts as zebrafish were treated with 1 µg/mL PTX. In contrast, the tumor sizes decreased in NCI-H23-TXR tumor xenografts with zebrafish pre-transfected with CS-PEI/Beclin-siRNA followed by the same treatment of PTX. The role of autophagy was associated with MDR development. This study paves the way for a new avenue of PTX in MDR-related lung cancer therapy using CS-PEI as a gene delivery carrier.

Targeting Tumor Microenvironment by Bioreduction-Activated Nanoparticles for Light-Triggered Virotherapy.

Solid tumors characteristically display higher levels of lactate production due to anaerobic metabolism of glucose. Meanwhile, the U.S. Food and Drug Administration (FDA) has approved virotherapy for use in cancer treatment; however systemic administration remains as a particular challenge. Here we report exploitation of tumor lactate production in designing a hypoxia-responsive carrier, self-assembled from hyaluronic acid (HA) conjugated with 6-(2-nitroimidazole)hexylamine, for localized release of recombinant adeno-associated virus serotype 2 (AAV2). The carrier is loaded with lactate oxidase (LOX) and is permeable to small molecules such as the lactate that accumulates in the tumor. Subsequently, LOX oxidizes the lactate to pyruvate inside the carrier, accompanied by internal lowering of oxygen partial pressure. Bioreduction of the 2-nitroimidazole of the HA conjugated with 6-(2-nitroimidazole)hexylamine converts it into a hydrophilic moiety and electrostatically dissociates the carrier and virus. Efficacious and specific delivery was proven by transduction of a photosensitive protein (KillerRed), enabling significant limitation in tumor growth in vivo with photodynamic therapy. An approximate 2.44-fold reduction in tumor weight was achieved after a 2-week course, compared with control groups. Furthermore, conjugation of the AAV2 with iron oxide nanoparticles ("magnetized" AAV2) facilitated magnetic resonance imaging tracking of the virus in vivo. Taken together, the solid tumor microenvironment promotes bioreduction of the lactate-responsive carrier, providing rapid and specific delivery of AAV2 for light-triggered virotherapy via systemic administration.

Light-triggered methylcellulose gold nanoparticle hydrogels for leptin release to inhibit fat stores in adipocytes.

Leptin is released in response to increased triglyceride storage in adipocytes and impacts body weight, but has drawbacks such as poor therapeutic effect and side effects when delivered systemically. Leptin also modifies adipocyte sensitivity to insulin to inhibit lipid accumulation. Here, light-triggered degradation of hydrogels was used to improve accuracy and effectiveness for sustained and controllable release. In our approach, leptin was entrapped within methylcellulose (MC)-based hydrogels, with incorporation of gold nanoparticles (NP). The incorporation of gold NP into MC hydrogels led to a tunable light irradiation response that dictated the hydrogel release rate of leptin. This manuscript demonstrates feasibility in designing tunable thermosensitive hydrogels for loading multimodality therapeutic agents to enhance the bioactivity of leptin for obesity therapy.

Magnetically Guided Viral Transduction of Gene-Based Sensitization for Localized Photodynamic Therapy To Overcome Multidrug Resistance in Breast Cancer Cells.

Chemotherapy represents a conventional treatment for many cancers at different stages and is either solely prescribed or concomitant to surgery, radiotherapy, or both. However, treatment is tempered in instances of acquired drug resistance in response to either chemotherapy or targeted therapy, leading to therapeutic failure. To overcome this challenge, many studies focus on how cancer cells manipulate their genomes and metabolism to prevent drug influx and facilitate the efflux of accumulated chemotherapy drugs. Herein, we demonstrate magnetic adeno-associated virus serotype 2 (ironized AAV2) has an ability to be magnetically guided and transduce the photosensitive KillerRed protein to enable photodynamic therapy irrespective of drug resistance.

Release of Doxorubicin by a Folate-Grafted, Chitosan-Coated Magnetic Nanoparticle.

In clinical tumor therapy, chemotherapeutic routes have caused severe side effects; current delivery methods are unsatisfactory. Successful design of a remotely folate (FA)-grafted chitosan (CS)-coated magnetic nanoparticle (MNP) with low toxicity, has been achieved. A chemotherapeutic drug such as doxorubicin (DOX), is loaded in the MNP-based matrix (FA-grafted CS-DOX-TPP-MNP), which is coated by an activated target tumor molecule of FA-grafted CS biopolymer with the inclusion of tripolyphosphate (TPP) as a linker. The resultant nano-complexes exhibited random aggregates (~240 nm) and zeta potential (-24.9 mV). In vivo experiments using athymic BALB/c nude mice with human glioblastoma U87 cells in a subcutaneous tumor model revealed that magnetic guidance of FA-grafted CS-DOX-TPP-MNP, injected via the tail vein, significantly decreased tumor growth. This manuscript demonstrates the feasibility of magnetizing control of FA-grafted CS-DOX-TPP-MNP to enhance the localization of drug release.

Remote Control of Light-Triggered Virotherapy.

Clinical virotherapy has been successfully approved for use in cancer treatment by the U.S. Food and Drug Administration; however, a number of improvements are still sought to more broadly develop virotherapy. A particular challenge is to administer viral therapy systemically and overcome limitations in intratumoral injection, especially for complex tumors within sensitive organs. To achieve this, however, a technique is required that delivers the virus to the tumor before the body's natural self-defense eradicates the virus prematurely. Here we show that recombinant adeno-associated virus serotype 2 (AAV2) chemically conjugated with iron oxide nanoparticles (∼5 nm) has a remarkable ability to be remotely guided under a magnetic field. Transduction is achieved with microscale precision. Furthermore, a gene for production of the photosensitive protein KillerRed was introduced into the AAV2 genome to enable photodynamic therapy (PDT), or light-triggered virotherapy. In vivo experiments revealed that magnetic guidance of "ironized" AAV2-KillerRed injected by tail vein in conjunction with PDT significantly decreases the tumor growth via apoptosis. This proof-of-principle demonstrates guided and highly localized microscale, light-triggered virotherapy.