Microneedle-assisted dual delivery of PUMA gene and celastrol for synergistic therapy of rheumatoid arthritis through restoring synovial homeostasis.

Abnormal proliferation of aggressive fibroblast-like synoviocytes (FLS) and perpetuate synovial inflammation can inevitably accelerate the progression of rheumatoid arthritis (RA). Herein, a strategy of simultaneously promoting FLS apoptosis and inhibiting inflammation as mediated by macrophages is proposed to restore synovial homeostasis for effective RA therapy. A hyaluronic acid-based dissolvable microneedle (MN) is fabricated for transdermal delivery of dual human serum albumin (HSA)-contained biomimetic nanocomplexes to regulate RA FLS and macrophages. Upon skin insertion, dual nanocomplexes are released rapidly from the MN and accumulate in RA joint microenvironment through both passive and active targeting as mediated by HSA. Thioketal-crosslinked fluorinated polyethyleneimine 1.8 K (TKPF) was constructed to bind the plasmid encoding pro-apoptotic gene PUMA with HSA coating layer (TKPF/pPUMA@HSA, TPH). TPH nanocomplexes can upregulate PUMA through RA FLS transfection to trigger efficient apoptosis. Also, HSA nanocomplexes encapsulating the classic anti-inflammatory natural product celastrol (Cel@HSA, CH) can inhibit inflammation of macrophages through blocking NF-κB pathway activation. TPH/CH MN can deplete RA FLS and inhibit M1 macrophage activation, suppress synovial hyperplasia as well as reduce bone and cartilage erosion in a collagen-induced arthritis (CIA) mouse model, demonstrating a promising strategy for efficient RA treatment.

GSH/pH Dual Activatable Cross-linked and Fluorinated PEI for Cancer Gene Therapy Through Endogenous Iron De-Hijacking and in Situ ROS Amplification.

Ferroptosis-related cancer therapy is limited by insufficient Fe2+ /Fe3+ redox pair and hydrogen peroxide (H2 O2 ) for producing lethal hydroxyl radicals (·OH). Although exogenous iron or ROS-producing drugs can enhance ferroptosis, exploiting endogenous iron (labile iron pool, LIP) stored in ferritin and promoting ROS generation may be safer. Herein, a metal/drug-free nanomedicine is developed for responsive LIP release and H2 O2 generation on the mitochondria membranes, amplifying hydroxyl radical production to enhance ferroptosis-mediated antitumor effects. A glutathione(GSH)/pH dual activatable fluorinated and cross-linked polyethyleneimine (PEI) with dialdehyde polyethylene glycol layer nanocomplex loaded with MTS-KR-SOD (Mitochondria-targeting-sequence-KillerRed-Superoxide Dismutase) and CRISPR/Cas9-CA IX (Carbonic anhydrase IX (CA IX)) plasmids (FP@MC) are developed for enhanced ferroptosis through endogenous iron de-hijacking and in situ ROS amplification. Two plasmids are constructed to knockdown CA IX and translate KillerRed-SOD recombinant protein specifically on mitochondria membranes, respectively. The CA IX knockdown acidifies the intracellular environment, leading the release of LIP from ferritin as a "flare" to initiate endogenous chemodynamic therapy. Meanwhile, MTS-KR-SOD generates H2 O2 when irradiated by a 590 nm laser to assist chemodynamic therapy, leading to ROS amplification for mitochondria damage and lipid peroxide accumulation. The combined therapeutic effects aggravate cancer ferroptosis and suppress tumor growth, providing a new paradigm for amplifying ROS and iron ions to promote ferroptosis-related cancer therapy.

Hierarchically tumor-activated nanoCRISPR-Cas13a facilitates efficient microRNA disruption for multi-pathway-mediated tumor suppression.

Rationale: CRISPR-Cas13a is an efficient tool for robust RNA knockdown with lower off-target effect, which may be a potentially powerful and safe tool for cancer gene therapy. However, therapeutic effect of current cancer gene therapy that targeting monogene was compromised by the multi-mutational signal pathway alterations of tumorigenesis. Methods: Here, hierarchically tumor-activated nanoCRISPR-Cas13a (CHAIN) is fabricated for multi-pathway-mediated tumor suppression by efficient microRNA disruption in vivo. A fluorinated polyetherimide (PEI; Mw=1.8KD) with graft rate of 33% (PF33) was utilized to compact the CRISPR-Cas13a megaplasmid targeting microRNA-21 (miR-21) (pCas13a-crRNA) via self-assemble to constitute a nanoscale 'core' (PF33/pCas13a-crRNA), which was further wrapped by modified hyaluronan (HA) derivatives (galactopyranoside-PEG2000-HA, GPH) to form CHAIN. Results: The dual-tumor-targeting and tumor-activated CHAIN not only manifested long-term circulation, but augmented tumor cellular uptake and endo/lysosomal escape, thus achieving efficient transfection of CRISPR-Cas13a megaplasmid (~ 13 kb) in tumor cells with minimal toxity. Efficient knockdown of miR-21 by CHAIN restored programmed cell death protein 4 (PDCD4) and reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) and further crippled downstream matrix metalloproteinases-2 (MMP-2), which undermined cancer proliferation, migration and invasion. Meanwhile, the miR-21-PDCD4-AP-1 positive feedback loop further functioned as an enhanced force for anti-tumor activity. Conclusion: Treatment with CHAIN in hepatocellular carcinoma mouse model achieved significant inhibition of miR-21 expression and rescued multi-pathway, which triggered substantial tumor growth suppression. By efficient CRISPR-Cas13a induced interference of one oncogenic microRNA, the CHAIN platform exerted promising capabilities in cancer treatment.

ROS-responsive fluorinated polyethyleneimine vector to co-deliver shMTHFD2 and shGPX4 plasmids induces ferroptosis and apoptosis for cancer therapy.

Ferroptosis is a newly discovered non-apoptotic cell death form but its therapeutic efficacy triggered by traditional iron-based nanomaterials or classic drug inducers has been far from satisfactory due to the high glutathione (GSH) level in cancer cells and insufficient lipid peroxide production. Here we reported a ferroptosis/apoptosis combinational therapy by depleting GSH and downregulating GPX4 to disrupt redox homeostasis and amplify ferroptosis-related oxidation effect. In this study, we developed reactive oxygen species (ROS)-responsive serum-resistant nanoparticles with thioketal-crosslinked fluorinated polyethyleneimine 1.8K (TKPF) as the core, which were wrapped with hyaluronic acid (HA) as the shell (TKPFH NP) to co-deliver shGPX4 and shMTHFD2 plasmids for cancer treatment. The highly efficient and tumor-selective gene carrier TKPFH NPs revealed outstanding transfection efficiency (∼100 %) and sustained the efficiency (∼50 %) even in media containing 90 % FBS. Mediated by HA, TKPFH NPs actively targeted CD44 receptors, thus enabling efficient uptake by tumor cells and experiencing surface charge conversion to induce subsequent lysosomal escape. Then the TKPF NPs were effectively disintegrated by the abundant ROS in cancer cells, which facilitated the release of plasmids and avoided the cytotoxicity of cationic polymers. shGPX4 plasmid induced ferroptosis by producing ROS and lipid peroxides via downregulating GPX4, while shMTHFD2 triggered apoptosis by modulating NADPH/NADP and depleting GSH of the cancer cells. Moreover, GSH consumption caused by shMTHFD2 indirectly suppressed GPX4 and further augmented ferroptosis, showing synergistic anticancer effect against B16-F10 cells. Taken together, the rationally designed dual-gene loaded TKPFH NPs provided a safe and high-performance platform for enhanced ferroptosis-apoptosis combined anticancer efficacy based on gene therapy. STATEMENT OF SIGNIFICANCE: The therapeutic efficacy of ferroptosis has been far from satisfactory due to high GSH level and insufficient lipid peroxide production in cancer cells. Herein, we reported a ferroptosis/apoptosis combinational therapy by depleting GSH and downregulating GPX4 to disrupt redox homeostasis and amplify ferroptosis-related oxidation effect. ROS-responsive serum-resistant nanoparticles were fabricated with thioketal-crosslinked fluorinated PEI 1.8K (TKPF) as the core and hyaluronic acid (HA) as the shell (TKPFH NP) to co-deliver shGPX4 and shMTHFD2 plasmids. The shGPX4 plasmid induced ferroptosis by producing ROS and lipid peroxides via downregulating GPX4, while shMTHFD2 triggered apoptosis by modulating NADPH/NADP and depleting GSH. The rationally designed dual-gene loaded TKPFH NPs provided a safe and high-performance platform aimed for enhanced ferroptosis-apoptosis combined anticancer efficacy.

Virus-esque nucleus-targeting nanoparticles deliver trojan plasmid for release of anti-tumor shuttle protein.

Gene therapy has gathered vast interest and been proved promising and prospective. While gene therapy evolves fast, demands of high transfecting efficiency and less toxic gene vectors are not sufficiently fulfilled. The progression of materials is doing the favor from which therapeutic application benefited is helping reshape treatments of cancer. In this work, we synthesized fluorinated branched polyethylenimine (PF33) and RGD-R8-PEG-HA (RRPH). When mixed with plasmids, the PF33 could form a compact nanoparticle PFC (Fluorinated PEI/plasmid Complex) and showed high transfection efficiency (>70% in A549 cells). Peptide modification and PEGylation on HA constituted the RRPH, and coating on the PFC would enable the ultimate nanoparticle RRPHC (RRPH coating PFC Complex) achieve long-term circulation and tumor tissue-penetration while maintaining the high transfection efficiency of PFC. Observations about the behavior in cellular organisms of RRPHC revealed its nucleus-targeting tendency. The in vivo distribution images revealed the RRPHC nanoparticles, compared to HAC (HA coated PFC, used as control) could achieve extended accumulation specifically on tumor regions rather than stay in other organs. While loaded with plasmids encoding our rationally designed trojan Apoptin (pSTA), RRPHC could establish compounds for the massive production of membrane-penetrating protein. Hence these cancer-killing proteins would charge at nucleus once phosphorylated and finish the task of destruction. Both in vitro and in vivo treatment using RRPHC/pSTA nanoparticles resulted in remarkable tumor suppression and the cytotoxicity tests demonstrated its low toxicity. In summary, pSTA encapsulating RRPHC nanoparticles may have potential applications in cancer gene therapy.

CRISPR-Cas9 Delivery by Artificial Virus (RRPHC).

Since its first harnessing in gene editing in 2012 and successful application in mammalian gene editing in 2013, the CRISPR-Cas9 system exerted magnificent power in all gene-editing-related applications, indicating a sharp thrive of this novel technology. However, there are still some critical drawbacks of the CRISPR-Cas9 system that hampered its broad application in gene editing. Efficient delivery of the Cas9 protein and its partner small guide RNA (sgRNA) to the target cells or tissue is one of the technical bottlenecks. CRISPR-Cas9 delivery via DNA plasmids still plays the big role in gene editing methods. With regard to the disadvantages of CRISPR-Cas9 plasmids, the most acute barrier lies in its large size (>10 kb) and the subsequent low transfection efficiency by conventional transfection method. In this chapter, what we present is an easy method by fabricating CRISPR-Cas9 plasmids into nanoparticle system and efficiently delivered into target cells to achieve gene editing.

An Endogenous Vaccine Based on Fluorophores and Multivalent Immunoadjuvants Regulates Tumor Micro-Environment for Synergistic Photothermal and Immunotherapy.

Recently, near-infrared (NIR) light-based photothermal therapy (PTT) has been widely applied in cancer treatment. However, in most cases, the tissue penetration depth of NIR light is not sufficient and thus photothermal therapy is unable to completely eradicate deep, seated tumors inevitably leading to recurrence of the tumor. Due to this significant limitation of NIR, improved therapeutic strategies are urgently needed. Methods: We developed an endogenous vaccine based on a novel nanoparticle platform for combinatorial photothermal ablation and immunotherapy. The design was based on fluorophore-loaded liposomes (IR-7-lipo) coated with a multivalent immunoadjuvant (HA-CpG). In vitro PTT potency was assessed in cells by LIVE/DEAD and Annexin V-FITC/PI assays. The effect on bone marrow-derived dendritic cells (BMDC) maturation and antigen presentation was evaluated by flow cytometry (FCM) with specific antibodies. After treatment, the immune cell populations in tumor micro-environment and the cytokines in the serum were detected by FCM and Elisa assay, respectively. Finally, the therapeutic outcome was investigated in an animal model. Results: Upon irradiation with 808 nm laser, IR-7-lipo induced tumor cell necrosis and released tumor-associated antigens, while the multivalent immunoadjuvant improved the expression of co-stimulatory molecules on BMDC and promoted antigen presentation. The combination therapy of PTT and immunotherapy regulated the tumor micro-environment, decreased immunosuppression, and potentiated host antitumor immunity. Most significantly, due to an enhanced antitumor immune response, combined photothermal immunotherapy was effective in eradicating tumors in mice and inhibiting tumor metastasis. Conclusion: This endogenous vaccination strategy based on synergistic photothermal and immunotherapy may provide a potentially effective approach for treatment of cancers, especially those difficult to be surgically removed.

Novel polyethyleneimine-R8-heparin nanogel for high-efficiency gene delivery in vitro and in vivo.

Gene therapy is an efficient and promising approach to treat malignant tumors. However, protecting the nucleic acid from degradation in vivo and efficient delivering it into tumor cells remain challenges that require to be addressed before gene therapy could be applied in clinic. In this study, we prepared novel polyethyleneimine-RRRRRRRR(R8)-heparin (HPR) nanogel as an efficient gene delivery system, which consists of heparin and cell penetrating peptide R8 grafted low-molecule-weight polyethyleneimine (PEI). Due to the shielding effect of heparin, crosslinking PEI-R8 with heparin was designed to diminish the toxicity of the gene delivery system. Meanwhile, a partial of R8 peptide which located on the surface of HPR nanogel could significantly enhance the cellular uptake. The formed HPR/pDNA complex exhibited effective endolysosomal escape, resulting in a high-efficiency transfection. Furthermore, the HPR could deliver the plasmid which could transcribe human TNF-related apoptosis inducing ligand (phTRAIL), into HCT-116 cells and induce significant cell apoptosis. In addition, HPR/phTRAIL complex showed satisfactory antitumor activity in abdominal metastatic colon carcinoma model. Finally, the antitumor mechanism of HPR/phTRAIL was also explored by western blot and histological analysis. The above results suggested that the HPR nanogel could serve as a promising gene delivery system.

Multifunctional Nucleus-targeting Nanoparticles with Ultra-high Gene Transfection Efficiency for In Vivo Gene Therapy.

Cancer stem cell-like cells (CSCL) are responsible for tumor recurrence associated with conventional therapy (e.g. surgery, radiation, and chemotherapy). Here, we developed a novel multifunctional nucleus-targeting nanoparticle-based gene delivery system which is capable of targeting and eradicating CSCL. These nanoparticles can facilitate efficient endosomal escape and spontaneously penetrate into nucleus without additional nuclear localization signal. They also induced extremely high gene transfection efficiency (>95%) even in culture medium containing 30% serum, which significantly surpassed that of some commercial transfection reagents, such as Lipofectamine 2000 and Lipofectamine 3000 etc. Especially, when loaded with the TRAIL gene, this system mediated remarkable depletion of CSCL. Upon systemic administration, the nanoparticles accumulated in tumor sites while sparing the non-cancer tissues and significantly inhibited the growth of tumors with no evident systemic toxicity. Taken together, our results suggest that these novel multifunctional, nucleus-targeting nanoparticles are a very promising in vivo gene delivery system capable of targeting CSCL and represent a new treatment candidate for improving the survival of cancer patients.

Biodegradable polymeric micelles coencapsulating paclitaxel and honokiol: a strategy for breast cancer therapy in vitro and in vivo.

The combination of chemotherapy drugs attracts more attention in clinical cancer trials. However, the poor water solubility of chemotherapeutic drugs restricts their anticancer application. In order to improve antitumor efficiency and reduce side effects of free drugs, we prepared paclitaxel (PTX) and honokiol (HK) combination methoxy poly(ethylene glycol)-poly(caprolactone) micelles (P-H/M) by solid dispersion method against breast cancer. The particle size of P-H/M was 28.7±2.5 nm, and transmission electron microscope image confirmed that P-H/M were spherical in shape with small particle size. After being encapsulated in micelles, the release of PTX or HK showed a sustained behavior in vitro. In addition, both the cytotoxicity and the cellular uptake of P-H/M were increased in 4T1 cells, and P-H/M induced more apoptosis than PTX-loaded micelles or HK-loaded micelles, as analyzed by flow cytometry assay and Western blot. Furthermore, the antitumor effect of P-H/M was significantly improved compared with PTX-loaded micelles or HK-loaded micelles in vivo. P-H/M were more effective in inhibiting tumor proliferation, inducing tumor apoptosis, and decreasing the density of microvasculature. Moreover, bioimaging analysis showed that drug-loaded polymeric micelles could accumulate more in tumor tissues compared with the free drug. Our results suggested that P-H/M may have potential applications in breast cancer therapy.

Artificial Virus Delivers CRISPR-Cas9 System for Genome Editing of Cells in Mice.

CRISPR-Cas9 has emerged as a versatile genome-editing platform. However, due to the large size of the commonly used CRISPR-Cas9 system, its effective delivery has been a challenge and limits its utility for basic research and therapeutic applications. Herein, a multifunctional nucleus-targeting "core-shell" artificial virus (RRPHC) was constructed for the delivery of CRISPR-Cas9 system. The artificial virus could efficiently load with the CRISPR-Cas9 system, accelerate the endosomal escape, and promote the penetration into the nucleus without additional nuclear-localization signal, thus enabling targeted gene disruption. Notably, the artificial virus is more efficient than SuperFect, Lipofectamine 2000, and Lipofectamine 3000. When loaded with a CRISPR-Cas9 plasmid, it induced higher targeted gene disruption efficacy than that of Lipofectamine 3000. Furthermore, the artificial virus effectively targets the ovarian cancer via dual-receptor-mediated endocytosis and had minimum side effects. When loaded with the Cas9-hMTH1 system targeting MTH1 gene, RRPHC showed effective disruption of MTH1 in vivo. This strategy could be adapted for delivering CRISPR-Cas9 plasmid or other functional nucleic acids in vivo.

Improving Antiadhesion Effect of Thermosensitive Hydrogel with Sustained Release of Tissue-type Plasminogen Activator in a Rat Repeated-Injury Model.

Intraperitoneal adhesion occurs frequently after pelvic and abdominal surgery, which plays an enormous burden on patients. Various drugs and barrier agents were studied and used to prevent adhesions, but few of them were satisfactory. A thermosensitive hydrogel was developed in our previous work and was effective in preventing adhesions. But in our preliminary experiment, it showed limited activity in a more rigorous rat repeated-injury adhesion model, which was much closer to clinical. In this study, tissue-type plasminogen activator (tPA) loaded thermosensitive hydrogel (tPA-hydrogel) was prepared, which combined barrier functions with sustained release of antiadhesion drug. The obtained tPA-hydrogel was injectable and degraded in vivo gradually in four weeks. Both hematoxylin and eosin and Masson trichrome staining confirmed that the tPA-hydrogel exhibited excellent antiadhesion effects on repeated-injury adhesion. Scanning electron microscopy was used to observe the injured abdominal wall, and cecum remesothelialized after treated with tPA-hydrogel for 14 d. In addition, the PAI-1 and tPA levels were measured by enzyme-linked immunosorbent assay. Results showed the PAI-1 concentrations in peritoneal lavage fluids of tPA-hydrogel treated rats were lower than that of other groups, leading to decreased fibrin formation, while there were no significant differences observed in tPA blood levels at any point in time (P > 0.05). This study demonstrated that the effectiveness of thermosensitive hydrogel in preventing adhesions could be enhanced by delivering antiadhesion drugs, and the tPA-hydrogel might be a promising system for clinical application.

Peritoneal adhesion prevention with a biodegradable and injectable N,O-carboxymethyl chitosan-aldehyde hyaluronic acid hydrogel in a rat repeated-injury model.

Postoperative peritoneal adhesion is one of the serious issues because it induces severe clinical disorders. In this study, we prepared biodegradable and injectable hydrogel composed of N,O-carboxymethyl chitosan (NOCC) and aldehyde hyaluronic acid (AHA), and assessed its anti-adhesion effect in a rigorous and severe recurrent adhesion model which is closer to clinical conditions. The flexible hydrogel, which gelated in 66 seconds at 37 °C, was cross-linked by the schiff base derived from the amino groups of NOCC and aldehyde groups in AHA. In vitro cytotoxicity test showed the hydrogel was non-toxic. In vitro and in vivo degradation examinations demonstrated the biodegradable and biocompatibility properties of the hydrogel. The hydrogel discs could prevent the invasion of fibroblasts, whereas fibroblasts encapsulated in the porous 3-dimensional hydrogels could grow and proliferate well. Furthermore, the hydrogel was applied to evaluate the anti-adhesion efficacy in a more rigorous recurrent adhesion model. Compared with normal saline group and commercial hyaluronic acid (HA) hydrogel, the NOCC-AHA hydrogel exhibited significant reduction of peritoneal adhesion. Compared to control group, the blood and abdominal lavage level of tPA was increased in NOCC-AHA hydrogel group. These findings suggested that NOCC-AHA hydrogel had a great potential to serve as an anti-adhesion candidate.

Multifunctional "core-shell" nanoparticles-based gene delivery for treatment of aggressive melanoma.

Gene therapy may be a promising and powerful strategy for cancer treatment, but efficient targeted gene delivery in vivo has so far remained challenging. Here, we developed a well-tailored and versatile "core-shell" ternary system (RRPHC) of systemic gene delivery for treatment of aggressive melanoma. The capsid-like "shell" of this system was engineered to mediate depth penetration to tissues, simultaneously target the CD44 receptors and integrin αvß3 receptors overexpressed on neovasculature and most malignant tumor cells, while the "core" was responsible for nucleus-targeting and effective transfection. The RRPHC ternary complexes enhanced cellular uptake via dual receptor-mediated endocytosis, improved the endosomal escape and significantly promoted the plasmid penetration into the nucleus. Notably, RRPHC ternary complexes exhibited ultra-high gene transfection efficiency (∼100% in B16F10 cells), which surpassed that of commercial transfection agents, PEI 25K, Lipofectamine 2000 and even Lipofectamine 3000. Especially, RRPHC ternary complexes showed excellent serum resistance and remained high gene transfection efficacy (∼100%) even in medium containing 30% serum. In vivo biodistribution imaging demonstrated RRPHC ternary complexes possessed much more accumulation and extensive distribution throughout tumor regions while minimal location in other organs. Furthermore, systemic delivery of the pro-apoptotic mTRAIL gene to tumor xenografts by RRPHC ternary complexes resulted in remarkable inhibition of melanoma, with no systemic toxicity. These results demonstrated that the designed novel RRPHC ternary complexes might be a promising gene delivery system for targeted cancer therapy in vivo.