Diabetic wounds are characterized by the disruption and cessation of essential healing stages, which include hemostasis, inflammation, proliferation, and remodeling. However, traditional treatments for diabetic wounds concentrate on individual stages of the healing process. Herein, this study utilizes mask-mediated sequential polymerization and varied cross-linking techniques to develop dual-modular microneedles (MNs) with fast- and slow-module, exhibiting varying degradation rates tailored for the full spectrum of diabetic wound healing. First, MNs incorporating calcium ions and dopamine synergistically promote rapid hemostasis. Second, fast-module physically cross-linked MNs rapidly D-mannose/dopamine-enhanced tripolyphosphate-quaternized chitosan (mDTC) nanoparticles (NPs) loaded with microRNA-147 (miRNA-147) to manage inflammation and oxidative stress in diabetic wounds. Additionally, dopamine in these NPs enhances their internalization and safeguards miRNA-147 from oxidative stress and RNase degradation. Finally, slow-module chemically cross-linked MNs facilitate the continuous release of deferoxamine (DFO) and dopamine, accelerating angiogenesis and tissue regeneration during the proliferation and remodeling stages. Manganese/dopamine-enhanced calcium peroxide NPs within the MNs initiate a blast-like generation of oxygen bubbles, not only enhancing the delivery of miRNA-mDTC NPs and DFO but also alleviating tissue hypoxia. Consequently, dual-modular MNs are instrumental in promoting rapid and complete healing of diabetic wounds through all stages of healing.

Lipid nanoparticles (LNPs) have successfully entered the clinic for the delivery of mRNA- and siRNA-based therapeutics, most recently as vaccines for COVID-19. Nevertheless, there is a lack of understanding regarding their in vivo behavior, in particular cell targeting. Part of this LNP tropism is based on the adherence of endogenous protein to the particle surface. This protein forms a so-called corona that can change, amongst other things, the circulation time, biodistribution and cellular uptake of these particles. The formation of this protein corona, in turn, is dependent on the nanoparticle properties (e.g., size, charge, surface chemistry and hydrophobicity) as well as the biological environment from which it is derived. With the potential of gene therapy to target virtually any disease, administration sites other than intravenous route are considered, resulting in tissue specific protein coronas. For neurological diseases, intracranial administration of LNPs results in a cerebral spinal fluid derived protein corona, possibly changing the properties of the lipid nanoparticle compared to intravenous administration. Here, the differences between plasma and CSF derived protein coronas on a clinically relevant LNP formulation were studied in vitro. Protein analysis showed that LNPs incubated in human CSF (C-LNPs) developed a protein corona composition that differed from that of LNPs incubated in plasma (P-LNPs). Lipoproteins as a whole, but in particular apolipoprotein E, represented a higher percentage of the total protein corona on C-LNPs than on P-LNPs. This resulted in improved cellular uptake of C-LNPs compared to P-LNPs, regardless of cell origin. Importantly, the higher LNP uptake did not directly translate into more efficient cargo delivery, underlining that further assessment of such mechanisms is necessary. These findings show that biofluid specific protein coronas alter LNP functionality, suggesting that the site of administration could affect LNP efficacy in vivo and needs to be considered during the development of the formulation.

Genome editing tools, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (e.g., CRISPR/Cas9), and their repurposing into epigenetic editing platforms, offer enormous potential as safe and customizable therapies for cancer. Specifically, various transcriptional abnormalities in human malignancies, such as silencing of tumor suppressors and ectopic re-expression of oncogenes, have been successfully targeted with virtually no off-target effects using CRISPR activation and repression systems. In these systems, the nuclease-deactivated Cas9 protein (dCas9) is fused to one or more domains inducing selective activation or repression of the targeted genes. Despite these advances, the efficient in vivo delivery of these molecules into the target cancer cells represents a critical barrier to accomplishing translation into a clinical therapy setting for cancer. Major obstacles include the large size of dCas9 fusion proteins, the necessity of multimodal delivery of protein and gRNAs, and the potential of these formulations to elicit detrimental immune responses.In this context, viral methods for delivering CRISPR face several limitations, such as the packaging capacity of the viral genome, the potential for integration of the nucleic acids into the host cells genome, and immunogenicity of viral proteins, posing serious safety concerns. The rapid development of mRNA vaccines in response to the COVID-19 pandemic has rekindled interest in mRNA-based approaches for CRISPR/dCas9 delivery. Simultaneously, due to their high loading capacity, scalability, customizable surface modification for cell targeting, and low immunogenicity, lipid nanoparticles (LNPs) have been widely explored as nonviral vectors. In this chapter, we first describe the design of optimized dCas9-effector mRNAs and gRNAs for epigenetic editing. We outline formulations of LNPs suitable for dCas9 mRNA delivery. Additionally, we provide a protocol for the co-encapsulation of the dCas9-effector mRNAs and gRNA into these LNPs, along with detailed methods for delivering these formulations to both cell lines (in vitro) and mouse models of breast cancer (in vivo).

Pancreatic cancer, predominantly pancreatic ductal adenocarcinoma (PDAC), remains a highly lethal malignancy with limited therapeutic options and a dismal prognosis. By targeting the underlying molecular abnormalities responsible for PDAC development and progression, gene therapy offers a promising strategy to overcome the challenges posed by conventional radiotherapy and chemotherapy. This study sought to explore the therapeutic potential of small activating RNAs (saRNAs) specifically targeting the CCAAT/enhancer-binding protein alpha (CEBPA) gene in PDAC. To overcome the challenges associated with saRNA delivery, tetrahedral framework nucleic acids (tFNAs) were rationally engineered as nanocarriers. These tFNAs were further functionalized with a truncated transferrin receptor aptamer (tTR14) to enhance targeting specificity for PDAC cells. The constructed tFNA-based saRNA formulation demonstrated exceptional stability, efficient saRNA release ability, substantial cellular uptake, biocompatibility, and nontoxicity. In vitro experiments revealed successful intracellular delivery of CEBPA-saRNA utilizing tTR14-decorated tFNA nanocarriers, resulting in significant activation of tumor suppressor genes, namely, CEBPA and its downstream effector P21, leading to notable inhibition of PDAC cell proliferation. Moreover, in a mouse model of PDAC, the tTR14-decorated tFNA-mediated delivery of CEBPA-saRNA effectively upregulated the expression of the CEBPA and P21 genes, consequently suppressing tumor growth. These compelling findings highlight the potential utility of saRNA delivered via a designed tFNA nanocarrier to induce the activation of tumor suppressor genes as an innovative therapeutic approach for PDAC.

BACKGROUND: Prostate cancer (PCa) has a high incidence in men worldwide, and almost all PCa patients progress to the androgen-independent stage which lacks effective treatment measures. PTENP1, a long non-coding RNA, has been shown to suppress tumor growth through the rescuing of PTEN expression via a competitive endogenous RNA (ceRNA) mechanism. However, PTENP1 was limited to be applied in the treatment of PCa for the reason of rapid enzymatic degradation, poor intracellular uptake, and excessively long base sequence to be synthesized. Considering the unique advantages of artificial nanomaterials in drug loading and transport, black phosphorus (BP) nanosheet was employed as a gene-drug carrier in this study.
RESULTS: The sequence of PTENP1 was adopted as a template which was randomly divided into four segments with a length of about 1000 nucleotide bases to synthesize four different RNA fragments as gene drugs, and loaded onto polyethyleneimine (PEI)-modified BP nanosheets to construct BP-PEI@RNA delivery platforms. The RNAs could be effectively delivered into PC3 cells by BP-PEI nanosheets and elevating PTEN expression by competitive binding microRNAs (miRNAs) which target PTEN mRNA, ultimately exerting anti-tumor effects.
CONCLUSIONS: Therefore, this study demonstrated that BP-PEI@RNAs is a promising gene therapeutic platform for PCa treatment.

The idea of mRNA therapy had been conceived for decades before it came into reality during the Covid-19 pandemic. The mRNA vaccine emerges as a powerful and general tool against new viral infections, largely due to its versatility and rapid development. In addition to prophylactic vaccines, mRNA technology also offers great promise for new applications as a versatile drug modality. However, realizing the conceptual potential faces considerable challenges, such as minimal immune stimulation, high and long-term expression, and efficient delivery to target cells and tissues. Here we review the applications of mRNA-based therapeutics, with emphasis on the innovative design and future challenges/solutions. In addition, we also discuss the next generation of mRNA therapy, including circular mRNA and self-amplifying RNAs. We aim to provide a conceptual overview and outlook on mRNA therapeutics beyond prophylactic vaccines.

Nucleic acid-based therapies are transforming medicine, but rely on an efficient delivery vehicle such as lipid nanoparticles (LNPs). Concerns exists in the nanomedicine field, that a large fraction of the LNPs in the ensemble does not contain any nucleic acid cargo and thus exert no functional effect. Nevertheless, how LNP lipid formulation, the LNP preparation method employed and nucleic acid cargo size correlates with the proportion of empty LNPs remains largely unexplored. Here we employ a well-established single particle based method to study nucleic acid loading heterogeneity in LNPs. We find that only a minor fraction of LNPs are \"empty\", both for LNPs loaded with siRNA, mRNA and plasmids. For clinically relevant LNPs for mRNA delivery, we never detected more than 16% empty nanoparticles in the ensemble. Thus employing standard LNP lipid-cargo combinations and preparation schemes results in LNPs with the potential to serve their biomedical function.

RNA-based therapeutics have emerged as a promising approach for the treatment of various diseases, including cancer, genetic disorders, and infectious diseases. However, the delivery of RNA molecules into target cells has been a major challenge due to their susceptibility to degradation and inefficient cellular uptake. To overcome these hurdles, DNA-based nano technology offers an unprecedented opportunity as a potential delivery platform for RNA therapeutics. Due to its excellent characteristics such as programmability and biocompatibility, these DNA-based nanostructures, composed of DNA molecules assembled into precise and programmable structures, have garnered significant attention as ideal building materials for protecting and delivering RNA payloads to the desired cellular destinations. In this review, we highlight the current progress in the design and application of three DNA-based nanostructures: DNA origami, lipid-nanoparticle (LNP) technology related to frame guided assembly (FGA), and DNA hydrogel for the delivery of RNA molecules. Their biomedical applications are briefly discussed and the challenges and future perspectives in this field are also highlighted.

Ribonucleic acid (RNA) therapeutics offer a broad prospect in cancer treatment. However, their successful application requires overcoming various physiological barriers to effectively deliver RNAs to the target sites. Currently, a number of RNA delivery systems based on polymeric nanoparticles are developed to overcome these barriers in RNA delivery. This work provides an overview of the existing RNA therapeutics for cancer gene therapy, and particularly summarizes those that are entering the clinical phase. This work then discusses the core features and latest research developments of tumor microenvironment-responsive polymer-based RNA delivery carriers which are designed based on the pathological characteristics of the tumor microenvironment. Finally, this work also proposes opportunities for the transformation of RNA therapies into cancer immunotherapy methods in clinical applications.

Transmembrane delivery of biologically active nucleic acids is an important process in cells and has inspired one to develop advanced drug delivery techniques. In this contribution, molecular-level single-stranded nucleic acid transmembrane carriers are reported based on 3.2 nm long Huc\'s foldamers (AOrnQ3Q3)8 and (mQ3Q2)8 with linearly and helically aligned positive charges, respectively. These two foldamers not only show very strong DNA affinity via electrostatic interactions but also discriminatively bind single-stranded DNA (ss-DNA) and double-stranded DNA (ds-DNA), corroborating the importance of precise charge arrangement in the electrostatic interactions. More importantly, these two foldamers are capable of efficiently transporting ss-DNA across the lipid membranes, and the ss-DNA transport activity of (AOrnQ3Q3)8 with linearly aligned charges is higher than that of (mQ3Q2)8 with helically aligned charges. Thus a type of novel single-stranded nucleic acid transmembrane molecular carriers based on positively charged helical foldamers are introduced. Further, effective and enhanced expression in EGFP-mRNA transfection experiments strongly demonstrates the potential of positively charged foldamers for RNA transmembrane transport and therapy.