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.

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.

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.

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.

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.

Highly specific in vivo gene delivery for p53-mediated apoptosis and genetic photodynamic therapies of tumour.

Anticancer therapies are often compromised by nonspecific effects and challenged by tumour environments' inherent physicochemical and biological characteristics. Often, therapeutic effect can be increased by addressing multiple parameters simultaneously. Here we report on exploiting extravasation due to inherent vascular leakiness for the delivery of a pH-sensitive polymer carrier. Tumours' acidic microenvironment instigates a charge reversal that promotes cellular internalization where endosomes destabilize and gene delivery is achieved. We assess our carrier with an aggressive non-small cell lung carcinoma (NSCLC) in vivo model and achieve >30% transfection efficiency via systemic delivery. Rejuvenation of the p53 apoptotic pathway as well as expression of KillerRed protein for sensitization in photodynamic therapy (PDT) is accomplished. A single administration greatly suppresses tumour growth and extends median animal survival from 28 days in control subjects to 68 days. The carrier has capacity for multiple payloads for greater therapeutic response where inter-individual variability can compromise efficacy.

Environment acidity triggers release of recombinant adeno-associated virus serotype 2 from a tunable matrix.

Successful design of a pH responsive polyelectrolyte-based virus delivery matrix with extracellular release triggered by tumor acidosis has been achieved. Recombinant adeno-associated virus serotype 2 (AAV2) is loaded in the polyelectrolyte-based matrix (AAV2-matrix), which is formed by a biodegradable copolymer of poly(polyethylene glycol-1-(3-aminopropyl)imidazole-dl-aspartic acid) with tuned pH response based on inclusion of polyethyleneimine (PEI(800)). Physico-chemical properties of AAV2-matrix are optimized to minimize cellular interactions until a tumor acidosis-like environment protonates the matrix, reverses ζ-potential and causes particles to swell, releasing the AAV2 virus. The pH-dependent release is highly controllable and potentially useful to optimize site specific viral delivery.

Extracellular delivery of modified oligonucleotide and superparamagnetic iron oxide nanoparticles from a degradable hydrogel triggered by tumor acidosis.

Chemically modified antisense RNA oligonucleotides (antagomir) offer promise for cancer therapies but suffer from poor therapeutic effect after systemic administration. Chemical modification or loading in degradable hydrogels can offer improvements in the accuracy and efficacy for sustained delivery at specific sites. In our approach, antagomir were entrapped with degradable poly(ethylene glycol) (PEG)-based hydrogels, with and without incorporation of imidazole. Superparamagnetic iron oxide nanoparticles (SPION) were simultaneously loaded with intent for magnetic resonance imaging (MRI). The incorporation of imidazole into the PEG hydrogels led to a tunable-pH-response that dictated hydrogel swelling ratio and release rate of antagomir and SPION. As a result, the PEG-imidazole hydrogel swelling ratio and degradation over a 5 week period changed up to 734% and 149% as the pH dropped from 7.4 to 6.7, respectively. The swelling ratio of PEG-imidazole hydrogels was completely reversible over repeatable cycles of pH change. The stimuli-responsive behavior of PEG-imidazole hydrogels was used for the release of antagomir and SPION under conditions consistent with tumor acidosis. This manuscript demonstrates feasibility in designing tunable-pH-responsive hydrogels for loading multimodality therapeutic and contrast agents to enhance the bioactivity of chemically modified antisense RNA oligonucleotide and SPION for acidosis-related tumor therapy and MRI imaging applications.

Switchable delivery of small interfering RNA using a negatively charged pH-responsive polyethylenimine-based polyelectrolyte complex.

A small interfering RNA (siRNA)-loaded polyelectrolyte constructed with branched polyethylenimine (bPEI) and copolymers, consisting of polyethylene glycol (PEG), histidine (His), and glutamic acid (Glu), was developed in order to provide a tumor acidosis-triggered delivery system with low cytotoxicity.

Controlled delivery of recombinant adeno-associated virus serotype 2 using pH-sensitive poly(ethylene glycol)-poly-L-histidine hydrogels.

Loading of viral vectors in synthetic polymers is a promising strategy for overcoming hurdles associated with viral gene delivery. For enhanced gene expression at a specific site, gene transfer by using hydrogels represents a versatile approach. In this study, adeno-associated virus serotype 2 containing the green fluorescent protein gene (rAAV2-GFP) were loaded into poly(ethylene glycol) (PEG) hydrogels, with and without incorporation of poly-L-hisditine (polyHis). Inclusion of polyHis created pH responsive hydrogels in a physiological range of tissues, containing the damaged vasculature and activated phagocytosis. The fraction of polyHis used controlled the degree of swelling, water uptake and subsequent degradation of the hydrogels and release rate of rAAV2-GFP. The swelling ratio of the PEG-polyHis hydrogels increased inversely with environment pH. As pH declined from 7.4 to 6.0, PEG-polyHis hydrogel swelling ratio and degradation rate increased 875% and 135%, respectively. As a result, release and transduction efficiency of the rAAV2-GFP from PEG-polyHis hydrogel in human HT-1080 fibrosarcoma cells increased significantly compared to a PEG hydrogel. Transduction rate can be controlled by the hydrogels' polyHis concentration and is sensitive to localized decreases in pH consistent with inflammation. This is relevant to optimizing parameters for wound care and regenerative medicine applications.

Electrostatic immobilization of DNA polyplexes on small intestinal submucosa for tissue substrate-mediated transfection.

Naturally occurring extracellular matrices (ECMs) such as small intestinal submucosa (SIS) have received significant attention for their therapeutic applications in tissue repair and regeneration. However, there have been no reports exploring the electrostatic properties of naturally occurring ECMs as a means to control transgene delivery. In the present study, we electrostatically adsorbed DNA polyplexes onto SIS for transfection upon cellular adhesion. To associate polyplexes with SIS, we first used a streaming potential method to characterize the surface charge of SIS and obtained a negative zeta potential at neutral pH, which can be attributed to the abundant glycosaminoglycan (GAG) content in SIS. We next prepared cationic polyethylenimine (PEI)/DNA polyplexes to associate with the negatively charged SIS for conjugation. Using the Cy(TM)3 dye-labeled control DNA as the reporter, we visualized the adsorption of PEI/DNA polyplexes at the SIS surface. Using luciferase, green fluorescent protein and beta-galactosidase as reporter proteins, we showed that the adsorbed PEI/DNA polyplexes were active and capable of carrying out transfection upon cellular adhesion, indicating that the electrostatic binding of polyplexes with SIS was reversible. In addition, the SIS-mediated transfection was contact-dependent: separation of SIS from the target cells via a 0.5 mm porous polyester membrane significantly reduced the efficiency of transfection in comparison to a direct seeding of cells onto SIS. We conclude that electrostatic immobilization of PEI/DNA polyplexes on SIS is capable of initiating efficient transgene delivery, which can be a useful tool in developing localized gene transfer.

Development of poly(amino ester glycol urethane)/siRNA polyplexes for gene silencing.

Nonviral vectors, with their low immunogenicity and lack of pathogenicity, offer significant promise for siRNA therapy with fewer safety concerns. Nonviral vectors were also preferred in most transient siRNA delivery due to their ease of preparation. Previously, we incorporated tertiary amines and polyethylene glycol (PEG) into poly(ester urethane) to synthesize a soluble poly(amino ester glycol urethane), PaE(G)U, as a novel DNA transfection reagent for transgene delivery. The aim of this study was to develop PaE(G)U/siRNA polyplexes for gene silencing. We characterized the properties of PaE(G)U/siRNA polyplexes and compared them with those of PaE(G)U/DNA polyplexes. Using the Alexa Fluor 488-labeled, nonsilencing control siRNA as the reporter, we visualized cellular uptake of PaE(G)U/siRNA polyplexes and optimized the mass ratio of PaE(G)U/siRNA for delivery at 80/1. At this ratio, the average diameter of polyplexes was 540 nm, which was significantly larger than the average diameter of PaE(G)U/DNA polyplexes at 155 nm for efficient DNA delivery. Using the optimized PaE(G)U/siRNA polyplexes, transient silencing of constitutive luciferase expression (up to 92%) was achieved in our recombinant human HT-1080 fibroblast model via anti-luciferase siRNA delivery. In conclusion, PaE(G)U/siRNA polyplexes were developed and optimized for cellular uptake to allow efficient gene silencing. Engineering of soluble biodegradable polymers to incorporate amino, ester, PEG, and urethane units in the backbone constitutes a useful approach for the future design of siRNA carriers.

EDC/NHS-mediated heparinization of small intestinal submucosa for recombinant adeno-associated virus serotype 2 binding and transduction.

A major challenge in the use of gene transfer vectors as therapeutic tools is controlling vector administration at a desired tissue site. One potential solution is implanting tissue-engineering constructs loaded with gene transfer vectors such as viruses for localized transgene delivery. In this work, we conjugated recombinant adeno-associated virus serotype 2 (rAAV2) to a heparinized small intestinal submucosa (H-SIS) matrix, which resulted in vector transduction upon cellular adhesion. H-SIS was prepared by incorporating heparin, the rAAV2 receptor, into SIS through N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) mediated crosslinking. Incorporated heparin adsorbed rAAV2 onto the H-SIS matrix for conjugation. Using green fluorescent protein and beta-galactosidase as reporters, we showed that conjugated rAAV2 was active and capable of mediating transgene delivery in cell culture. Additionally, we applied H-SIS to adsorb unpurified rAAV2 from the crude lysate of packaging cells for conjugation, avoiding the use of ultracentrifugation or chromatography in preparation of infectious rAAV2 for transduction. Our work provides a unique, modified tissue substrate H-SIS for rAAV2 binding and transduction, which can be a useful tool in developing localized gene transfer.