Metabolic engineering of commensal bacteria for gut butyrate delivery and dissection of host-microbe interaction.

An overwhelming number of studies have reported the correlation of decreased abundance of butyrate-producing commensals with a wide range of diseases. However, the molecular-level mechanisms whereby gut butyrate causally affects the host mucosal immunity and pathogenesis were poorly understood, hindered by the lack of efficient tools to control intestinal butyrate. Here we engineered a facultative anaerobic commensal bacterium to delivery butyrate at the intestinal mucosal surface, and implemented it to dissect the causal role of gut butyrate in regulating host intestinal homeostasis in a model of murine chronic colitis. Mechanistically, we show that gut butyrate protected against colitis and preserved intestinal mucosal homeostasis through its inhibiting effect on the key pyroptosis executioner gasdermin D (GSDMD) of colonic epithelium, via functioning as an HDAC3 inhibitor. Overall, our work presents a new avenue to build synthetic living delivery bacteria to decode causal molecules at the host-microbe interface with molecular-level insights.

Endogenous dual miRNA-triggered dynamic assembly of DNA nanostructures for in-situ dual siRNA delivery.

A theranostic strategy of multiple microRNA (miRNA)-triggered in-situ delivery of small interfering RNA (siRNA) can effectively improve the precise therapy of cancer cells. Benefiting from the advantages of programmability, specific molecular recognition, easy functionalization and marked biocompatibility of DNA nanostructures, we designed a three-dimensional (3D) DNA nano-therapeutic platform for dual miRNA-triggered in-situ delivery of siRNA. The 3D DNA nanostructure (TY1Y2) was constructed based on the self-assembly of a DNA tetrahedra scaffold, two sets of Y-shaped DNA (Y1 and Y2), and EpCAM-aptamer which functionalized as the ligand molecule for the recognition of specific cancer cells. After being specifically internalized into the targeted cancer cells, TY1Y2 was triggered by two endogenous miRNAs (miR-21 and miR-122), resulting in the generation of strong fluorescence resonance energy transfer fluorescent signal for dual miRNAs imaging. Meanwhile, the therapeutic siRNAs (siSurvivin and siBcl2) could also be in-situ generated and released from TY1Y2 through the strand-displacement reactions for the synergistic gene therapy of cancer cells. This 3D DNA nanostructure integrated the specific imaging of endogenous biomarkers and the in-situ delivery of therapeutic genes into the multifunctional nanoplatform, revealing the promising applications for the diagnosis and treatment of cancer. Electronic Supplementary Material: Supplementary material is available in the online version of this article at 10.1007/s40843-022-2420-y.

Sandwich-Structured Implants to Obstruct Multipath Energy Supply and Trigger Self-Enhanced Hypoxia-Initiated Chemotherapy Against Postsurgical Tumor Recurrence and Metastasis.

As a currently common strategy to treat cancer, surgical resection may cause tumor recurrence and metastasis due to residual postoperative tumors. Herein, an implantable sandwich-structured dual-drug depot is developed to trigger a self-intensified starvation therapy and hypoxia-induced chemotherapy sequentially. The two outer layers are 3D-printed using a calcium-crosslinked mixture ink containing soy protein isolate, polyvinyl alcohol, sodium alginate, and combretastatin A4 phosphate (CA4P). The inner layer is one patch of poly (lactic-co-glycolic acid)-based electrospun fibers loaded with tirapazamine (TPZ). The preferentially released CA4P destroys the preexisting blood vessels and prevents neovascularization, which obstructs the external energy supply to cancer cells but aggravates hypoxic condition. The subsequently released TPZ is bioreduced to cytotoxic benzotriazinyl under hypoxia, further damaging DNA, generating reactive oxygen species, disrupting mitochondria, and downregulating hypoxia-inducible factor 1α, vascular endothelial growth factor, and matrix metalloproteinase 9. Together these processes induce apoptosis, block the intracellular energy supply, counteract the disadvantage of CA4P in favoring intratumor angiogenesis, and suppress tumor metastasis. The in vivo and in vitro results and the transcriptome analysis demonstrate that the postsurgical adjuvant treatment with the dual-drug-loaded sandwich-like implants efficiently inhibits tumor recurrence and metastasis, showing great potential for clinical translation.

Comparison ofin-situversusex-situdelivery of polyethylenimine-BMP-2 polyplexes for rat calvarial defect repair via intraoperative bioprinting.

Gene therapeutic applications combined with bio- and nano-materials have been used to address current shortcomings in bone tissue engineering due to their feasibility, safety and potential capability for clinical translation. Delivery of non-viral vectors can be altered using gene-activated matrices to improve their efficacy to repair bone defects.Ex-situandin-situdelivery strategies are the most used methods for bone therapy, which have never been directly compared for their potency to repair critical-sized bone defects. In this regard, we first time explore the delivery of polyethylenimine (PEI) complexed plasmid DNA encoding bone morphogenetic protein-2 (PEI-pBMP-2) using the two delivery strategies,ex-situandin-situdelivery. To realize these gene delivery strategies, we employed intraoperative bioprinting (IOB), enabling us to 3D bioprint bone tissue constructs directly into defect sites in a surgical setting. Here, we demonstrated IOB of an osteogenic bioink loaded with PEI-pBMP-2 for thein-situdelivery approach, and PEI-pBMP-2 transfected rat bone marrow mesenchymal stem cells laden bioink for theex-situdelivery approach as alternative delivery strategies. We found thatin-situdelivery of PEI-pBMP-2 significantly improved bone tissue formation compared toex-situdelivery. Despite debates amongst individual advantages and disadvantages ofex-situandin-situdelivery strategies, our results ruled in favor of thein-situdelivery strategy, which could be desirable to use for future clinical applications.

Controlled Co-delivery of pPDGF-B and pBMP-2 from intraoperatively bioprinted bone constructs improves the repair of calvarial defects in rats.

Intraoperative bioprinting (IOB), which refers to the bioprinting process performed on a live subject in a surgical setting, has made it feasible to directly deliver gene-activated matrices into craniomaxillofacial (CMF) defect sites. In this study, we demonstrated a novel approach to overcome the current limitations of traditionally fabricated non-viral gene delivery systems through direct IOB of bone constructs into defect sites. We used a controlled co-delivery release of growth factors from a gene-activated matrix (an osteogenic bioink loaded with plasmid-DNAs (pDNA)) to promote bone repair. The controlled co-delivery approach was achieved from the combination of platelet-derived growth factor-B encoded plasmid-DNA (pPDGF-B) and chitosan-nanoparticle encapsulating pDNA encoded with bone morphogenetic protein-2 (CS-NPs(pBMP2)), which facilitated a burst release of pPDGF-B in 10 days, and a sustained release of pBMP-2 for 5 weeks in vitro. The controlled co-delivery approach was tested for its potential to repair critical-sized rat calvarial defects. The controlled-released pDNAs from the intraoperatively bioprinted bone constructs resulted in ∼40% bone tissue formation and ∼90% bone coverage area at 6 weeks compared to ∼10% new bone tissue and ∼25% total bone coverage area in empty defects. The delivery of growth factors incorporated within the intraoperatively bioprinted constructs could pose as an effective way to enhance bone regeneration in patients with cranial injuries in the future.

Engineered implantable vaccine platform for continuous antigen-specific immunomodulation.

Cancer vaccines harness the host immune system to generate antigen-specific antitumor immunity for long-term tumor elimination with durable immunomodulation. Commonly investigated strategies reintroduce ex vivo autologous dendritic cells (DCs) but have limited clinical adoption due to difficulty in manufacturing, delivery and low clinical efficacy. To combat this, we designed the "NanoLymph", an implantable subcutaneous device for antigen-specific antitumor immunomodulation. The NanoLymph consists of a dual-reservoir platform for sustained release of immune stimulants via a nanoporous membrane and hydrogel-encapsulated antigens for local immune cell recruitment and activation, respectively. Here, we present the development and characterization of the NanoLymph as well as efficacy validation for immunomodulation in an immunocompetent murine model. Specifically, we established the NanoLymph biocompatibility and mechanical stability. Further, we demonstrated minimally invasive transcutaneous refilling of the drug reservoir in vivo for prolonging drug release duration. Importantly, our study demonstrated that local elution of two drugs (GMCSF and Resiquimod) generates an immune stimulatory microenvironment capable of local DC recruitment and activation and generation of antigen-specific T lymphocytes within 14 days. In summary, the NanoLymph approach can achieve in situ immunomodulation, presenting a viable strategy for therapeutic cancer vaccines.

Emerging biomaterial-based strategies for personalized therapeutic in situ cancer vaccines.

Landmark successes in oncoimmunology have led to development of therapeutics boosting the host immune system to eradicate local and distant tumors with impactful tumor reduction in a subset of patients. However, current immunotherapy modalities often demonstrate limited success when involving immunologically cold tumors and solid tumors. Here, we describe the role of various biomaterials to formulate cancer vaccines as a form of cancer immunotherapy, seeking to utilize the host immune system to activate and expand tumor-specific T cells. Biomaterial-based cancer vaccines enhance the cancer-immunity cycle by harnessing cellular recruitment and activation against tumor-specific antigens. In this review, we discuss biomaterial-based vaccine strategies to induce lymphocytic responses necessary to mediate anti-tumor immunity. We focus on strategies that selectively attract dendritic cells via immunostimulatory gradients, activate them against presented tumor-specific antigens, and induce effective cross-presentation to T cells in secondary lymphoid organs, thereby generating immunity. We posit that personalized cancer vaccines are promising targets to generate long-term systemic immunity against patient- and tumor-specific antigens to ensure long-term cancer remission.

Emerging technologies for local cancer treatment.

The fundamental limitations of systemic therapeutic administration have prompted the development of local drug delivery platforms as a solution to increase effectiveness and reduce side effects. By confining therapeutics to the site of disease, local delivery technologies can enhance therapeutic index. This review highlights recent advances and opportunities in local drug delivery strategies for cancer treatment in addition to challenges that need to be addressed to facilitate clinical translation. The benefits of local cancer treatment combined with technological advancements and increased understanding of the tumor microenvironment, present a prime breakthrough opportunity for safer and more effective therapies.

Delivery of Nanoparticle-Based Radiosensitizers for Radiotherapy Applications.

Nanoparticle-based radiosensitization of cancerous cells is evolving as a favorable modality for enhancing radiotherapeutic ratio, and as an effective tool for increasing the outcome of concomitant chemoradiotherapy. Nevertheless, delivery of sufficient concentrations of nanoparticles (NPs) or nanoparticle-based radiosensitizers (NBRs) to the targeted tumor without or with limited systemic side effects on healthy tissues/organs remains a challenge that many investigators continue to explore. With current systemic intravenous delivery of a drug, even targeted nanoparticles with great prospect of reaching targeted distant tumor sites, only a portion of the administered NPs/drug dosage can reach the tumor, despite the enhanced permeability and retention (EPR) effect. The rest of the targeted NPs/drug remain in systemic circulation, resulting in systemic toxicity, which can decrease the general health of patients. However, the dose from ionizing radiation is generally delivered across normal tissues to the tumor cells (especially external beam radiotherapy), which limits dose escalation, making radiotherapy (RT) somewhat unsafe for some diseased sites despite the emerging development in RT equipment and technologies. Since radiation cannot discriminate healthy tissue from diseased tissue, the radiation doses delivered across healthy tissues (even with nanoparticles delivered via systemic administration) are likely to increase injury to normal tissues by accelerating DNA damage, thereby creating free radicals that can result in secondary tumors. As a result, other delivery routes, such as inhalation of nanoparticles (for lung cancers), localized delivery via intratumoral injection, and implants loaded with nanoparticles for local radiosensitization, have been studied. Herein, we review the current NP delivery techniques; precise systemic delivery (injection/infusion and inhalation), and localized delivery (intratumoral injection and local implants) of NBRs/NPs. The current challenges, opportunities, and future prospects for delivery of nanoparticle-based radiosensitizers are also discussed.

Hydrogel-Assisted Antisense LNA Gapmer Delivery for In Situ Gene Silencing in Spinal Cord Injury.

After spinal cord injury (SCI), nerve regeneration is severely hampered due to the establishment of a highly inhibitory microenvironment at the injury site, through the contribution of multiple factors. The potential of antisense oligonucleotides (AONs) to modify gene expression at different levels, allowing the regulation of cell survival and cell function, together with the availability of chemically modified nucleic acids with favorable biopharmaceutical properties, make AONs an attractive tool for novel SCI therapy developments. In this work, we explored the potential of locked nucleic acid (LNA)-modified AON gapmers in combination with a fibrin hydrogel bridging material to induce gene silencing in situ at a SCI lesion site. LNA gapmers were effectively developed against two promising gene targets aiming at enhancing axonal regeneration-RhoA and GSK3ß. The fibrin-matrix-assisted AON delivery system mediated potent RNA knockdown in vitro in a dorsal root ganglion explant culture system and in vivo at a SCI lesion site, achieving around 75% downregulation 5 days after hydrogel injection. Our results show that local implantation of a AON-gapmer-loaded hydrogel matrix mediated efficient gene silencing in the lesioned spinal cord and is an innovative platform that can potentially combine gene regulation with regenerative permissive substrates aiming at SCI therapeutics and nerve regeneration.

In situ delivery of corticosteroids for treatment of oral diseases.

In many mucocutaneous disorders, corticosteroids therapy is currently central. Systemic therapy is restricted to severe disorders whereas topical applications are considered as the first-line treatment. The oral cavity environment, the medication form and other factors related to the delivery method are key factors for the therapy efficiency and effectiveness. Current marketed medications are not able to avoid wrong drug exposure and scarce patients' compliance. Innovative in situ delivery systems are able to prolong the drug retention time on the mucosa and to avoid the drawbacks of conventional formulations. This review is intended to give a general overview of oral mucocutaneous pathologies and highlight the potential of new technologies in designing innovative delivery systems able to release corticosteroids in situ for the treatment of various oral cavity disorders.

Tumor-selective lipopolyplex encapsulated small active RNA hampers colorectal cancer growth in vitro and in orthotopic murine.

Small active RNA (saRNA)-induced gene activation (RNAa) is a novel strategy to treat cancer. Our previous work proved that the p21-saRNA-322 successfully hindered colorectal cancer growth by activating p21 gene. However, the barrier for successful saRNA therapy is lack of efficient drug delivery. In the present study, a rectal delivery system entitled p21-saRNA-322 encapsulated tumor-selective lipopolyplex (TSLPP-p21-saRNA-322) which consist of PEI/p21-saRNA-322 polyplex core and hyaluronan (HA) modulated lipid shell was developed to treat colorectal cancer. Our results showed that this system maintained at the rectum for more than 6 h and preferentially accumulated at tumor site. CD44 knock down experiment instructed that the superb cellular uptake of TSLPP-p21-saRNA-322 attributed to HA-CD44 recognition. An orthotopic model of bio-luminescence human colorectal cancer in mice was developed using microsurgery and TSLPP-p21-saRNA-322 demonstrated a superior antitumor efficacy in vitro and in vivo. Our results provide preclinical proof-of-concept for a novel method to treat colorectal cancer by rectal administration of TSLPP formulated p21-saRNA-322.

Nanoparticulate platforms for targeting bone infections: meeting a major therapeutic challenge.

Bone infections are devastating complications in orthopedics due to biofilm formation. Treatment requires high antibiotic doses, which may lead to systemic toxicity thus limiting the drug therapeutic effectiveness. In this context, nanoparticles are well-known controlled release drug carriers that are able to modulate release rate, versatile in terms of administration routes and may be used as local delivery systems. Regarding bone infections, although nanoparticles are a promising strategy for overcoming biofilm tolerance, there are clearly technical, safety, regulatory and clinical challenges that need to be overcome before such nanomedicines may be translated into clinical use. In this paper, we present a critical overview on the high expectations against the real potential of the nanotechnological approaches to bone infection treatment.