Non-viral vector-based genome editing for cancer immunotherapy.

Despite the exciting promise of cancer immunotherapy in the clinic, immune checkpoint blockade therapy and T cell-based therapies are often associated with low response rates, intrinsic and adaptive immune resistance, and systemic side effects. CRISPR-Cas-based genome editing appears to be an effective strategy to overcome these unmet clinical needs. As a safer delivery platform for the CRISPR-Cas system, non-viral nanoformulations have been recently explored to target tumor cells and immune cells, aiming to improve cancer immunotherapy on a gene level. In this review, we summarized the efforts of non-viral vector-based CRISPR-Cas-mediated genome editing in tumor cells and immune cells for cancer immunotherapy. Their design rationale and specific applications were highlighted.

Trehalose enhanced cold atmospheric plasma-mediated cancer treatment.

Cold atmospheric plasma (CAP) is a unique form of physical plasma that has shown great potential for cancer therapy. CAP uses ionized gas to induce lethal oxidative stress on cancer cells; however, the efficacy of CAP therapy continues to be improved. Here, we report an injectable hydrogel-mediated approach to enhance the anti-tumor efficacy of CAP by regulating the phosphorylation of eIF2α. We discovered that reactive oxygen and nitrogen species (ROS/RNS), two main anti-tumor components in CAP, can lead to lethal oxidative stress on tumor cells. Elevated oxidative stress subsequently induces eIF2α phosphorylation, a pathognomonic marker of immunogenic cell death (ICD). Trehalose, a natural disaccharide sugar, can further enhance CAP-induced ICD by elevating the phosphorylation of eIF2α. Moreover, injectable hydrogel-mediated delivery of CAP/trehalose treatment promoted dendritic cell (DC) maturation, initiating tumor-specific T-cell mediated anti-tumor immune responses. The combination therapy also supported the polarization of tumor-associated macrophages to an M1-like phenotype, reversing the immunosuppressive tumor microenvironment and promoting tumor antigen presentation to T cells. In combination with immune checkpoint inhibitors (i.e., anti-programmed cell death protein 1 antibody, aPD1), CAP/trehalose therapy further inhibited tumor growth. Importantly, our findings also indicated that this hydrogel-mediated local combination therapy engaged the host systemic innate and adaptive immune systems to impair the growth of distant tumors.

Cotargeting CDK4/6 and BRD4 Promotes Senescence and Ferroptosis Sensitivity in Cancer.

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are approved for breast cancer treatment and show activity against other malignancies, including KRAS-mutant non-small cell lung cancer (NSCLC). However, the clinical efficacy of CDK4/6 inhibitors is limited due to frequent drug resistance and their largely cytostatic effects. Through a genome-wide cDNA screen, we identified that bromodomain-containing protein 4 (BRD4) overexpression conferred resistance to the CDK4/6 inhibitor palbociclib in KRAS-mutant NSCLC cells. Inhibition of BRD4, either by RNA interference or small-molecule inhibitors, synergized with palbociclib to induce senescence in NSCLC cells and tumors, and the combination prolonged survival in a KRAS-mutant NSCLC mouse model. Mechanistically, BRD4-inhibition enhanced cell-cycle arrest and reactive oxygen species (ROS) accumulation, both of which are necessary for senescence induction; this in turn elevated GPX4, a peroxidase that suppresses ROS-triggered ferroptosis. Consequently, GPX4 inhibitor treatment selectively induced ferroptotic cell death in the senescent cancer cells, resulting in tumor regression. Cotargeting CDK4/6 and BRD4 also promoted senescence and ferroptosis vulnerability in pancreatic and breast cancer cells. Together, these findings reveal therapeutic vulnerabilities and effective combinations to enhance the clinical utility of CDK4/6 inhibitors. SIGNIFICANCE: The combination of cytostatic CDK4/6 and BRD4 inhibitors induces senescent cancer cells that are primed for activation of ferroptotic cell death by targeting GPX4, providing an effective strategy for treating cancer.

Engineered Nanomaterials to Potentiate CRISPR/Cas9 Gene Editing for Cancer Therapy.

Clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) gene-editing technology shows promise for manipulating single or multiple tumor-associated genes and engineering immune cells to treat cancers. Currently, most gene-editing strategies rely on viral delivery; yet, while being efficient, many limitations, mainly from safety and packaging capacity considerations, hinder the use of viral CRISPR vectors in cancer therapy. In contrast, recent advances in non-viral CRISPR/Cas9 nanoformulations have paved the way for better cancer gene editing, as these nanoformulations can be engineered to improve safety, efficiency, and specificity through optimizing the packaging capacity, pharmacokinetics, and targetability. In this review, the advance in non-viral CRISPR delivery is highlighted, and there is a discussion on how these approaches can be potentially used to treat cancers in addressing the aforementioned limitations, followed by the perspectives in designing a proper CRISPR/Cas9-based cancer nanomedicine system with translational potential.

Injectable cold atmospheric plasma-activated immunotherapeutic hydrogel for enhanced cancer treatment.

Despite the promise of immune checkpoint blockade (ICB) for cancer treatment, challenges associated with this therapy still exist, including low response rates and severe side effects in patients. Here, we report a hydrogel-mediated combination therapy for enhanced ICB therapy. Specifically, cold atmospheric plasma (CAP), an ionized gas consisting of therapeutically effective reactive oxygen species (ROS) and reactive nitrogen species (RNS), can effectively induce cancer immunogenic cell death, releasing tumor-associated antigens in situ and initiating anti-tumor immune responses, which, therefore, can synergistically augment the efficacy of immune checkpoint inhibitors. To minimize the systemic toxicity of immune checkpoint inhibitors and improve the tissue penetration of CAP, an injectable Pluronic hydrogel was employed as a delivery method. Our results show that major long-lived ROS and RNS in CAP can be effectively persevered in Pluronic hydrogel and remain efficacious in inducing cancer immunogenic cell death after intratumoral injection. Our findings suggest that local hydrogel-mediated combination of CAP and ICB treatment can evoke both strong innate and adaptive, local and systemic anti-tumor immune responses, thereby inhibiting both tumor growth and potential metastatic spread.

Supramolecular biomaterials for enhanced cancer immunotherapy.

Cancer immunotherapy has achieved promising clinical results. However, many limitations associated with current cancer immunotherapy still exist, including low response rates and severe adverse effects in patients. Engineering biomaterials for the delivery of immunotherapeutic reagents has been suggested to be an effective strategy to improve cancer immunotherapy. Among different biomaterials, supramolecular biomaterials with flexible and versatile structures and functions have exhibited unparalleled advantages in promoting cancer immunotherapy. In recent years, various supramolecular formulations have been extensively explored as immunotherapeutic delivery platforms due to their high cargo-loading capacity/feasibility, facile immunization function, and excellent biocompatibility, which make them possible candidates for modular and personalized cancer immunotherapy. These nanoarchitectures with unique topologies possess distinguishing advantages in cancer immunotherapy, incarnating a structure-property relationship. Based on extensive state-of-the-art research, this minireview highlights recent advances in supramolecular biomaterials for cancer immunotherapy and discusses the possible mechanisms underlying how supramolecular biomaterials promote the development of cancer immunotherapy together with their potential for clinical translation.

Reactive oxygen species / photothermal therapy dual-triggered biomimetic gold nanocages nanoplatform for combination cancer therapy via ferroptosis and tumor-associated macrophage repolarization mechanism.

With the continuous development of cancer nanotechnology, an important trend in the research is to combine the broad application prospects of functional nanomaterials with recent biological discoveries and technological advances. Herein, a cancer cell membrane-camouflaged gold nanocage loading doxorubicin (DOX) and l-buthionine sulfoximine (BSO) (denoted as m@Au-D/B NCs) was constructed as an innovative nanoplatform to confer promising cancer combination therapy by evoking effective ferroptosis and immune responses. Briefly, the loading of BSO and DOX could induce ferroptosis through simultaneous effective glutathione (GSH) consumption and reactive oxygen species (ROS) accumulation. Gold nanocages (AuNCs) with distinct anti-tumor application performance was utilized as ideal nanocarrier for drug loading, evoking photothermal effects and photochemical catalysis to generate more ROS under near-infrared (NIR) irradiation. Moreover, m@Au-D/B NCs-mediated photothermal therapy (PTT) combined with ROS production could repolarize the tumor-associated macrophages (TAMs) from pro-tumor (M2) phenotype to anti-tumor (M1) phenotype, thus improving tumor-suppressive immune environment and then promoting the activation of effector cells and release of pro-inflammatory cytokines, in which the antitumor responses were evoked robustly in a methodical approach. The anti-tumor effects in vivo implied that m@Au-D/B NCs could significantly inhibit tumor growth without severe toxicity. Hence, this homotypic targeting nanosystem could offer an auspicious anticancer access by triggering combination cancer therapy via ferroptosis and tumor-associated macrophage repolarization mechanism.

pH-Sensitive Molecular-Switch-Containing Polymer Nanoparticle for Breast Cancer Therapy with Ferritinophagy-Cascade Ferroptosis and Tumor Immune Activation.

Ferritin internalized into tumor cells is degraded and releases iron ions via ferritinophagy. Iron ions participate in Fenton reaction to produce reactive oxygen species for lipid peroxidation and ferroptosis. Inhibition of indoleamine-2,3-dioxygenase (IDO) decreases tryptophan elimination to induce T cells activation for tumor immunosuppression relief. The active tumor targeting nanoparticles containing ferritin and a pH-sensitive molecular-switch (FPBC@SN) are developed to utilize ferritinophagy-cascade ferroptosis and tumor immunity activation for cancer therapy. FPBC@SN disintegrates in acidic cytoplasm and releases sorafenib (SRF) and IDO inhibitor (NLG919). SRF upregulates nuclear receptor coactivator 4 (NCOA4) to induce ferritin and endogenous iron pool degradation by ferritinophagy, then obtained iron ions participate in the Fenton reaction to produce lipid peroxide (LPO). Meanwhile, SRF blocks glutathione synthesis to downregulate glutathione peroxidase 4 (GPX4) which can scavenge LPO as a different pathway from ferritinophagy to promote ferroptosis in tumor cells. NLG919 inhibits IDO to reduce tryptophan metabolism, so immunity in tumors is aroused to anti-tumor. In vitro and in vivo experiments prove FPBC@SN inhibits tumor cell growth and metastasis, indicating the potential of FPBC@SN for breast cancer therapy based on the combination of ferritinophagy-cascade ferroptosis and tumor immunity activation.

Multifunctional ZnO@CuS nanoparticles cluster synergize chemotherapy and photothermal therapy for tumor metastasis.

The poor drug delivery and unsatisfying therapeutic effects remain to be the primary challenges for cancer therapy. Nanosystem that combines multiple functions into a single platform is an ideal strategy. Here, a smart drug delivery nanoplatform (Z@C-D/P) based on ZnO@CuS nanoparticles, loaded with doxorubicin (DOX) and pirfenidone (PFD) was constructed. Importantly, the ß-CD-DMA and PEG-DMA could be activated in the mild acidic tumor microenvironment, then the nanosystem underwent charge reversal and PFD release. PFD could inhibit cancer-associated fibroblasts (CAFs) activation and enhance tumor penetration. And the residual nanostructure ZnO@CuS could trigger cascade amplified ROS generation to induce tumor cell death. The photothermal effect further strengthened the anti-tumor efficacy. Finally, the nanosystem showed remarkable inhibition of tumor growth (89.7%) and lung metastasis. The innovatively designed nanosystem integrating chemotherapy and photothermal effect would provide a promising strategy in breast cancer therapy.

Dual-Responsive and Deep-Penetrating Nanomicelles for Tumor Therapy via Extracellular Matrix Degradation and Oxidative Stress.

Tumor microenvironment (TME), with complex composition, plays a vital role in the occurrence, development, and metastasis of tumors. TME becomes an important obstacle to the accessibility of nanotherapy, thus indicating the need to improve the functional design to overcome this challenge. In this study, we generate an intelligent nano-drug-delivery system (DOX@PssP-Hh NPs) with dual environmental response, which involves heparanase (HPSE) in TME and glutathione (GSH) in tumor cells. The nanosystem consists of a nanoskeleton formed by self-assembly of mPEG-ss-PEI and α-CD (PssP), chemotherapy drug doxorubicin (DOX) for enhancing antitumor efficacy, together with hyaluronidase (HAase), which is designed to degrade extracellular matrix to increase drug penetration, and an outer shell of heparin. Through the process of "responsive disintegration-remodeling tumor microenvironment-enhancing drug penetration-inducing oxidative stress", the semi-rotaxaneself-assembled nanomicelles were constructed to achieve the progressive function. DOX@PssP-Hh NPs with the size of 81.85 ± 1.85 nm exhibited satisfactory cytotoxicity (IC50 = 0.80 ± 0.33 µg/mL). With the disulfide bond-mediated GSH depletion and DOX-mediated reactive oxygen species (ROS) production, treatment with DOX@PssP-Hh NPs prominently reduced glutathione peroxidase 4 (GPX4) level and would lead to enhanced oxidative stresses. Hyaluronic acid (HA), collagen I, and α-smooth muscle actin (α-SMA) were significantly reduced for TME remodulation. Moreover, the antitumor effect in vivo implied that DOX@PssP-Hh NPs could inhibit tumor growth effectively and reduce tumor interstitial fluid pressure (IFP) evidently. In conclusion, DOX@PssP-Hh NPs improved the penetration of drugs and exhibited enhanced antitumor efficacy.

Chemo-Photothermal Combination Cancer Therapy with ROS Scavenging, Extracellular Matrix Depletion, and Tumor Immune Activation by Telmisartan and Diselenide-Paclitaxel Prodrug Loaded Nanoparticles.

Extracellular matrix (ECM) accumulating in the tumor microenvironment (TME) is generated by tumor-associated fibroblasts. It can elevate interstitial fluid pressure and form dense barriers in tumor tissues. Consequently, nanocarriers are hindered from permeating into deeper tumor sites. Thus, the programmed drug-releasing nanoparticles, G(TM)PPSP, were developed for TME remodeling and breast cancer therapy. Gelatin nanoparticles were linked with platinum nanoparticles (PtNPs) to obtain G(TM)PPSP with a size of 214.0 ± 5.0 nm. Telmisartan (TM) was loaded in gelatin nanoparticles. Paclitaxel (PTX) was attached to PtNPs via a dual redox responsive diselenide bond. TM release was mediated by MMP-2 because of gelatin degradation in TME, and then intracellular PTX was released because of diselenide linkage fracture triggered by ROS or glutathione. ECM was depleted owing to TGF-ß downregulation by TM and direct ablation by the photothermal effect of PtNPs. 4T1 tumor progression was inhibited by PTX chemotherapy, intracellular ROS scavenging of PtNPs, and photothermal therapy (PTT). The tumor spheroid penetration assay proved G(TM)PPSP could permeate into deep tumor regions when MMP-2 existed. In vivo antitumor experiments implied G(TM)PPSP with PTT could inhibit tumor growth effectively and remodel TME via ECM depletion and immunity activation, indicating the potential of G(TM)PPSP-based chemo-photothermal combination therapy for breast cancer treatment.

Fenton-reaction-stimulative nanoparticles decorated with a reactive-oxygen-species (ROS)-responsive molecular switch for ROS amplification and triple negative breast cancer therapy.

Triple-negative breast cancer (TNBC) is characterized by a high metastatic rate, which can seriously threaten women's health. ROS play an important role in tumor development and metastasis. Excessive ROS can induce tumor cell apoptosis and inhibit tumor cell metastasis. This study investigated Fenton-reaction-stimulative nanoparticles (P@P/H NPs) containing ROS-responsive molecular switches for antitumor metastasis by amplifying the ROS and activating the cascade biological reaction of ROS in tumor cells. Spheroidal P@P/H NPs exhibited a uniform size of 68.18 ± 0.29 nm, high drug cumulative release of 97.59% in response to H2O2 at 24 h, and satisfactory cytotoxicity with the IC50 value of 0.50 ± 0.02 µg mL-1. The markedly elevated ROS level caused by P@P/H NPs generated an evident antitumor metastasis effect in vitro by facilitating the expressions of cytochrome c, caspase-9, and caspase-3 and blocking that of matrix metalloprotein 9 (MMP-9). Moreover, P@P/H NPs engendered an excellent tumor inhibition rate of 56.37% and antitumor metastasis effect in vivo. Therefore, P@P/H NPs could respond to H2O2 in tumor cells to rapidly disassemble and further increase the ROS to induce antitumor metastasis via the Fenton reaction.

Preparation of Deoxycholate-Modified Docetaxel-Cimetidine Complex Chitosan Nanoparticles to Improve Oral Bioavailability.

Docetaxel (DTX) was effective in the treatment of neoplasm but could only be administered intravenously with the poor oral bioavailability owing to its undesirable solubility, remarkably metabolic conversion, and other factors. Cimetidine (CMD), a classic CYP3A4 isozyme inhibitor, had exhibited a wide range of inhibition on the metabolism of many drugs. The aim of this study was to construct the novel docetaxel-cimetidine (DTX-CMD) complex and the chitosan-deoxycholate nanoparticles based on it to confirm whether this formulation could show advantages in terms of solubility, dissolution rate, small intestinal absorption, and oral bioavailability in comparison with the pure drug. The solid-state characterization was carried out by powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FT-IR), and simultaneous DSC-TGA (SDT). Dissolution rate and kinetic solubility study were determined by evaluating the amount of DTX in distilled water and phosphate buffer solution (pH = 7.4), respectively. And small intestinal absorption and pharmacokinetics study were conducted in rats. The results of this study demonstrated that we successfully constructed DTX-CMD complex and its chitosan-deoxycholate nanoparticles. Furthermore, the DTX-CMD complex increased the solubility of DTX by 2.3-fold and 2.1-fold in distilled water and phosphate buffer solution, respectively. The ultimate accumulative amount of DTX-CMD complex nanoparticles through rat small intestinal in 2 h was approximately 4.9-fold and the oral bioavailability of the novel nanoparticles was enhanced 2.8-fold, compared with the pure DTX. The superior properties of the complex nanoparticles could both improve oral bioavailability and provide much more feasibility for other formulations of DTX.