Advances in the synthesis and applications of macrocyclic polyamines.

Macrocyclic polyamines constitute a significant class of macrocyclic compounds that play a pivotal role in the realm of supramolecular chemistry. They find extensive applications across diverse domains including industrial and agricultural production, clinical diagnostics, environmental protection and other multidisciplinary fields. Macrocyclic polyamines possess a distinctive cavity structure with varying sizes, depths, electron-richness degrees and flexibilities. This unique feature enables them to form specific supramolecular structures through complexation with diverse objects, thereby attracting considerable attention from chemists, biologists and materials scientists alike. However, there is currently a lack of comprehensive summaries on the synthesis methods for macrocyclic polyamines. In this review article, we provide an in-depth introduction to the synthesis of macrocyclic polyamines while analysing their respective advantages and disadvantages. Furthermore, we also present an overview of the recent 5-year advancements in using macrocyclic polyamines as non-viral gene vectors, fluorescent probes, diagnostic and therapeutic reagents as well as catalysts. Looking ahead to future research directions on the synthesis and application of macrocyclic polyamines across various fields will hopefully inspire new ideas for their synthesis and use.

Recent advances in the development of poly(ester amide)s-based carriers for drug delivery.

Biodegradable and biocompatible biomaterials have several important applications in drug delivery. The biomaterial family known as poly(ester amide)s (PEAs) has garnered considerable interest because it exhibits the benefits of both polyester and polyamide, as well as production from readily available raw ingredients and sophisticated synthesis techniques. Specifically, α-amino acid-based PEAs (AA-PEAs) are promising carriers because of their structural flexibility, biocompatibility, and biodegradability. Herein, we summarize the latest applications of PEAs in drug delivery systems, including antitumor, gene therapy, and protein drugs, and discuss the prospects of drug delivery based on PEAs, which provides a reference for designing safe and efficient drug delivery carriers.

Feasibility of Direct Vitrectomy-Sparing Subretinal Injection for Gene Delivery in Large Animals.

PURPOSE: To assess the safety and feasibility of direct vitrectomy-sparing subretinal injection for gene delivery in a large animal model. METHODS: The experimental Libechov minipigs were used for subretinal delivery of a plasmid DNA vector (pS/MAR-CMV-copGFP) with cytomegalovirus (CMV) promoter, green fluorescent protein (GFP) reporter (copGFP) and a scaffold/matrix attachment region (S/MAR) sequence. The eyes were randomized to subretinal injection of the vector following pars plana vitrectomy (control group) or a direct injection without prior vitrectomy surgery (experimental group). Intra- and post-operative observations up to 30 days after surgery were compared. RESULTS: Six eyes of three mini-pigs underwent surgery for delivery into the subretinal space. Two eyes in the control group were operated with a classical approach (lens-sparing vitrectomy and posterior hyaloid detachment). The other four eyes in the experimental group were injected directly with a subretinal cannula without vitrectomy surgery. No adverse events, such as endophthalmitis, retinal detachment and intraocular pressure elevation were observed post-operatively. The eyes in the experimental group had both shorter surgical time and recovery while achieving the same surgical goal. CONCLUSIONS: This pilot study demonstrates that successful subretinal delivery of gene therapy vectors is achievable using a direct injection without prior vitrectomy surgery.

Tuning the Potency of Farnesol-Modified Polyethylenimine with Polyanionic Trans-Booster to Enhance DNA Delivery.

Low molecular weight polyethylenimine (PEI) based lipopolymers become an attractive strategy to construct nonviral therapeutic carriers with promising transfection efficiency and minimal toxicity. Herein, this paper presents the design and synthesis of novel farnesol (Far) conjugated PEI, namely PEI1.2k-SA-Far7. The polymers had quick DNA complexation, effective DNA unpacking (dissociation), and cellular uptake abilities when complexed with plasmid DNA. However, they were unable to provide robust transfection in culture, indicating inability of Far grafting to improve the transfection efficacy significantly. To overcome this limitation, the commercially available polyanionic Trans-Booster additive, which is capable of displaying electrostatic interaction with PEI1.2k-SA-Far7, has been used to enhance the uptake of pDNA polyplexes and transgene expression. pDNA condensation was successfully achieved in the presence of the Trans-Booster with more stable polyplexes, and in vitro transfection efficacy of the polyplexes was improved to be comparable to that obtained with an established reference reagent. The PEI1.2k-SA-Far7/pDNA/Trans-Booster ternary complex exhibited good compatibility with cells and minimal hemolysis activity. This work demonstrates the exemplary potency of using additives in polyplexes and the potential of resultant ternary complexes for effective pDNA delivery.

PEI functionalized cell membrane for tumor targeted and glutathione responsive gene delivery.

Polyethylenimine (PEI) is a broadly exploited cationic polymer due to its remarkable gene-loading capacity. However, the high cytotoxicity caused by its high surface charge density has been reported in many cell lines, limiting its application significantly. In this study, two different molecular weights of PEI (PEI10k and PEI25k) were crosslinked with red blood cell membranes (RBCm) via disulfide bonds to form PEI derivatives (RMPs) with lower charge density. Furthermore, the targeting molecule folic acid (FA) molecules were further grafted onto the polymers to obtain FA-modified PEI-RBCm copolymers (FA-RMP25k) with tumor cell targeting and glutathione response. In vitro experiments showed that the FA-RMP25k/DNA complex had satisfactory uptake efficiency in both HeLa and 293T cells, and did not cause significant cytotoxicity. Furthermore, the uptake and transfection efficiency of the FA-RMP25k/DNA complex was significantly higher than that of the PEI25k/DNA complex, indicating that FA grafting can increase transfection efficiency by 15 %. These results suggest that FA-RMP25k may be a promising non-viral gene vector with potential applications in gene therapy.

PEGylated pH-responsive peptide-mRNA nano self-assemblies enhance the pulmonary delivery efficiency and safety of aerosolized mRNA.

Inhalable messenger RNA (mRNA) has demonstrated great potential in therapy and vaccine development to confront various lung diseases. However, few gene vectors could overcome the airway mucus and intracellular barriers for successful pulmonary mRNA delivery. Apart from the low pulmonary gene delivery efficiency, nonnegligible toxicity is another common problem that impedes the clinical application of many non-viral vectors. PEGylated cationic peptide-based mRNA delivery vector is a prospective approach to enhance the pulmonary delivery efficacy and safety of aerosolized mRNA by oral inhalation administration. In this study, different lengths of hydrophilic PEG chains were covalently linked to an amphiphilic, water-soluble pH-responsive peptide, and the peptide/mRNA nano self-assemblies were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The in vitro mRNA binding and release, cellular uptake, transfection, and cytotoxicity were studied, and finally, a proper PEGylated peptide with enhanced pulmonary mRNA delivery efficiency and improved safety in mice was identified. These results showed that a proper N-terminus PEGylation strategy using 12-monomer linear monodisperse PEG could significantly improve the mRNA transfection efficiency and biocompatibility of the non-PEGylated cationic peptide carrier, while a longer PEG chain modification adversely decreased the cellular uptake and transfection on A549 and HepG2 cells, emphasizing the importance of a proper PEG chain length selection. Moreover, the optimized PEGylated peptide showed a significantly enhanced mRNA pulmonary delivery efficiency and ameliorated safety profiles over the non-PEGylated peptide and LipofectamineTM 2000 in mice. Our results reveal that the PEGylated peptide could be a promising mRNA delivery vector candidate for inhaled mRNA vaccines and therapeutic applications for the prevention and treatment of different respiratory diseases in the future.

Enhancing the antitumor immunosurveillance of PD-L1-targeted gene therapy for metastatic melanoma using cationized Panax Notoginseng polysaccharide.

Improved curative effects with reduced toxicity has always been the ultimate goal of gene delivery vectors for tumor immunotherapy. Panax notoginseng polysaccharide (PNP), a natural plant-derived macromolecule, not only has antitumor immune activity but also has the typical structural characteristics useful for gene delivery. In this work, positively charged polyethyleneimine (PEI) was directly grafted to the backbone of PNP to induced its charge reversal and generate a functional gene vector (PNP-PEI). Moreover, a short hairpin RNA targeting the programmed death-ligand 1 (PD-L1) was loaded into PNP-PEI to generate a potentially therapeutic nanoparticle (PNP-PEI/shPD-L1). In vitro and in vivo experiments demonstrated that PNP-PEI could efficiently carry the therapeutic shPD-L1 into tumor cells and that PNP-PEI/shPD-L1 could significantly inhibit the expression of PD-L1 and growth of B16-F10 cells. Noteworthily, treatment with PNP-PEI reversed the phenotype of macrophages from M2 to M1 subtype and promoted dendritic cell maturation, which encouraged the host immunity and enhanced the therapeutic antitumor effects. In summary, this study describes a PNP-based gene delivery vector and highlights the beneficial immunopotentiating therapeutic outcomes of PNP-PEI for tumor immunotherapy.

AAV-Mediated Somatic Gene Editing for Cardiac and Skeletal Muscle in a Large Animal Model.

Here we describe a protocol to produce a recombinant adeno-associated viral vector (rAAV)-based system to deliver the CRISPR-Cas9 complex into porcine skeletal muscle and myocardial cells. We initially describe the genomic composition of the rAAV-CRISPR vectors used in our lab. Furthermore, we give a step-by-step instruction into the production of recombinant viral vectors with high yields and purity. Lastly we describe the minimally invasive injection regimes to target the myocardium in a pig.

A simple pre-disease state prediction method based on variations of gene vector features.

BACKGROUND: The progression of disease can be divided into three states: normal, pre-disease, and disease. Since a pre-disease state is the tipping point of disease deterioration, accurately predicting pre-disease state may help to prevent the progression of disease and develop feasible treatment in time. METHODS: In the perspective of gene regulatory network, the expression of a gene is regulated by its upstream genes, and then it also regulates that of its downstream genes. In this study, we define the expression value of these genes as a gene vector to depict its state in a specific sample. Then, we propose a novel pre-disease prediction method by such vector features. RESULTS: The results of an influenza virus infection dataset show that our method can successfully predict the pre-disease state. Furthermore, the pre-disease state related genes predicted by our methods are highly associated with each other and enriched in influenza virus infection related pathways. In addition, our method is more time efficient in calculation than previous works. The code of our method is accessed at https://github.com/ZhenshenBao/sPGVF.git.

Polysaccharides: A Carrier for Gene Therapy.

Recently, polysaccharides have been proved to be an interesting drug delivery system as gene carriers, and natural polysaccharides have attracted more attention in the field of biomaterials due to their unique non-toxicity, good biocompatibility, and biodegradability. Compared with other polysaccharides, chitosan and dextran have more extensive and profound applications and can directly or indirectly deliver therapeutic genes. Chinese medicinal polysaccharides often do not have cationic groups and cannot directly deliver therapeutic genes. They usually need certain chemical modifications. The operation process is difficult, but better therapeutic effects are achieved. In this paper, the classification of polysaccharides and the research progress in modified polysaccharides as novel gene carriers are reviewed to provide a reference for polysaccharides as novel gene carriers in gene therapy. Moreover, efficient transfection of DNA and RNA therapeutic genes requires the development of new efficient and low-toxic vectors as well as the optimization of existing delivery vectors. Therefore, the research on polysaccharide gene vector has a long way to go and has good prospects in scientific research and biomedicine.

Watson-Crick Base Pairing-Inspired Laser/GSH Activatable miRNA-Coordination Polymer Nanoplexes for Combined Cancer Chemo-Immuno-Photothermal Therapy.

The tumor immunosuppressive microenvironment (TIM) greatly hindered the efficacy of cancer immunotherapy. Overexpressed indoleamine 2,3-dioxygenase-1 (IDO1) in tumor tissues plays a vital role in TIM generation, and downregulation of IDO1 expression may reverse TIM. Inspired by the Watson-Crick base-pairing rule, a versatile noncationic miRNA vector (miDAC@PDA) is developed for cancer immunotherapy. Doxorubicin (DOX), adenosine triphosphate (ATP), and copper ions (Cu2+) are coassembled into coordination polymer nanoparticles (DAC) and bind miRNA via the hydrogen bond interaction (miDAC) between adenine residues (ATP) and uracil residues (miRNA). Polydopamine (PDA) is deposited onto the surface of miDAC for photothermal therapy. miDAC@PDA can efficiently accumulate into tumor tissues for cellular uptake. Under laser irradiation and high intracellular GSH levels, the PDA shell of miDAC@PDA can dissociate from miDAC for miRNA release due to local hyperthermia. Cu2+-mediated GSH consumption and intracellular ATP release can amplify the DOX-based immunogenic cell death (ICD) cascade, together with miR-448-mediated IDO1 inhibition, and these versatile nanoplexes will not only restrain primary tumor growth but also display a remarkable abscopal effect on distant tumors. Collectively, our study provides a unique strategy for intracellular gene delivery and an inspirational approach for multimechanism cancer management.

Mechanistic Studies and Formulation Mitigations of Adeno-associated Virus Capsid Rupture During Freezing and Thawing: Mechanisms of Freeze/Thaw Induced AAV Rupture.

Gene therapies delivered using adeno-associated virus (AAV) vectors are showing promise for many diseases. Frozen AAV drug products are exposed to freeze-thaw (F/T) cycles during manufacturing, storage, and distribution. In this work we studied the mechanisms of AAV capsid rupture during F/T. We found that exposure to interfaces, exacerbated by F/T, and the mechanical force of excipient devitrification correlated with AAV capsid rupture during F/T. There was no impact of pH shifts, cryo-concentration, or cold-denaturation. Results were similar for AAV8 and AAV9. With these mechanistic insights we identified three formulation mitigation approaches. Addition of ≥0.0005% w/v poloxamer 188 (P188) eliminated substantial recovery losses (up to ∼60% without P188) and minimized rupture to ≤1% per F/T cycle. Elimination of exothermic devitrification events during rewarming, either by formulating with a low buffer concentration, or by adding a cryoprotectant further reduced rupture during F/T. Rupture of AAV9 was <0.2% per F/T cycle in a formulation with 1 mM phosphate, 4.4 mM dextrose, electrolytes, and 0.001% P188 at pH 7.2. Rupture of AAV8 was not detected when formulated with 4% sucrose, 100 mM salt, and 0.001% P188 at pH 7.4. These results provide insights into effective strategies for stabilizing AAVs against rupture during F/T.

Systematic Studies on Stabilization of AAV Vector Formulations by Lyophilization.

Adeno-associated virus (AAV) vectors have evolved as one of the most promising delivery systems for gene therapy. The current standard for AAV vector storage is deep-freezing below -60 °C. While this allows for long-term vector storage without loss of activity, it is inconvenient and involves high costs and logistical challenges. Therefore, there is a need for AAV formulations, such as freeze-dried formulations, that allow for long-term storage at 2-8 °C. A major challenge in developing a lyophilization process for complex biological structures like an AAV vector is to minimize the stress on the capsid during the lyophilization cycle. Here, we evaluated different conditions for freeze-drying of AAV8 vectors and found that undesirable instability can be significantly reduced if secondary drying is performed at lower temperatures, kept as short as possible, and the residual moisture is kept between 1.5 and 2%. In a next step, we explored formulations with different salt concentration or excipient compositions and found that a combination of 10 mM phosphate buffer, 5.67% (150 mM) trehalose, 5% hydroxyectoine and 0.1% poloxamer with a residual moisture of approx. 1.5% provided stable long-term storage at 2-8 °C and for at least 4 weeks at 25 °C. These results pave the way for future optimizations of freeze-drying processes for AAV vector-based gene therapy products.

Hyaluronic acid-modified redox-sensitive hybrid nanocomplex loading with siRNA for non-small-cell lung carcinoma therapy.

A novel hyaluronic acid (HA)-modified hybrid nanocomplex HA-SeSe-COOH/siR-93C@PAMAM, which could efficiently deliver siRNA into tumor cells via a redox-mediated intracellular disassembly, was constructed for enhanced antitumor efficacy. Thereinto, siR-93C (siRNA) and positive PAMAM were firstly mixed into the electrostatic nano-intermediate, and then diselenide bond (-SeSe-)-modified HA was coved to shield excessive positive charges. This hybrid nanocomplex displayed uniform dynamic sizes, high stability, controlled zeta potential and narrow PDI distribution. Moreover, the -SeSe- linkage displayed GSH/ROS dual responsive properties, improving intracellular trafficking of siRNA. In vitro assays in A549 cell line presented that HA-SeSe-COOH/siR-93C@PAMAM has low cytotoxicity, rapid lysosomal escape and significant transfection efficiency; besides, an efficient proliferation inhibition ability and enhanced apoptosis. Furthermore, in animal studies, this negative-surfaced hybrid nanocomplex showed a prolonged circulation in blood and improved inhibition of tumor growth. All these results verified our hypothesis in this study that diselenide bonds-modified HA could promote not only stability and safety of nanoparticles in vivo but also intracellular behavior of siRNA via redox-dual sensitive properties; furthermore, this hybrid nanocomplex provided a visible potential approach for siRNA delivery in the antitumor field.

Impact of Time Out of Intended Storage and Freeze-thaw Rates on the Stability of Adeno-associated Virus 8 and 9.

There are an increasing number of clinical studies evaluating different adeno-associated virus (AAV) serotypes as vectors for gene therapy. Long-term frozen storage can maximize the stability of AAV. Freeze-thaw (F/T) cycles and exposures to room temperature (RT) and refrigerated conditions occur during manufacturing, labeling, and clinical use. In this work we exposed AAV8 and AAV9 at low and high concentrations to five F/T cycles compounded with RT and refrigerated holds in a 'daisy chain' time out of intended storage (TOIS) stability study, which may be a best practice in early development. We also evaluated the impact of 5 F/T cycles for multiple permutations of fast and slow cooling and rewarming rates. The quality attributes of AAV8 and AAV9 remained within acceptable ranges after the daisy chain TOIS and F/T rate studies. Potency and concentration were unchanged within method variability. There was a minor increase in non-encapsidated ('free') DNA released from AAV8 after F/T in a phosphate-buffered saline formulation. DNA release during F/T was minimized in a formulation with a low buffer concentration and was not detected in a formulation containing sucrose. We conclude that AAV8 and AAV9 have stability profiles that are suitable for manufacturing and clinical development.

Surface Coating of Pulmonary siRNA Delivery Vectors Enabling Mucus Penetration, Cell Targeting, and Intracellular Radical Scavenging for Enhanced Acute Lung Injury Therapy.

Pulmonary delivery of anti-inflammatory siRNA presents a promising approach for localized therapy of acute lung injury (ALI), while polycationic vectors can be easily trapped by the negatively charged airway mucin glycoproteins and arbitrarily internalized by epithelial cells with nontargetability for immunological clearance. Herein, we report a material, the dopamine (DA)-grafted hyaluronic acid (HA-DA), coating on an anti-TNF-α vector to address these limitations. HA-DA was simply synthesized and facilely coated on poly(ß-amino ester) (BP)-based siRNA vectors via electrostatic attraction. The resulting HA-DA/BP/siRNA displayed significantly enhanced mucus penetration, attributable to the charge screen effect of HA-DA and the bioadhesive nature of the grafting DA. After transmucosal delivery, the nanosystem could target diseased macrophages via CD44-mediated internalization and rapidly escape from endo/lysosomes through the proton sponge effect, resulting in effective TNF-α regulation. Meanwhile, DA modification endowed the coating material with robust antioxidative capability to scavenge a broad spectrum of reactive oxygen/nitrogen species (RONS), which protected the lung tissue from oxidative damage and synergized with anti-TNF-α to inhibit a cytokine storm. As a result, a remarkable amelioration of ALI was achieved in a lipopolysaccharide (LPS)-stimulated mice model. This study provides a multifunctional coating material to facilitate pulmonary drug delivery for the treatment of lung diseases.

Zinc(II)-Cyclen Multifunctional Complex Module-Mediated Polycation-Based High-Performance pDNA Vectors.

A kind of novel multifunctional modules based on zinc(II)-coordinative cyclen has been developed, which is utilized to modify low molecular weight polyethylenimine (LMWPEI) obtaining high-performance DNA vectors. A series of in vitro experiments were carried out to explore the performance of the module in improving the key process of gene transfection, such as DNA condensation, serum resistance, cellular uptake, and endosomal escape. The results demonstrate that there is a significant synergistic effect between the functional module and PEI2.5k in the process of breaking through the key barriers of gene transfection. The optimal Zn-PCD mediates 160-fold higher gluciferase activity than commercial transfection reagents PEI25k in ADSC stem cells with more than 90% cell viability and achieves excellent transfection efficiency in diverse cell types, for instance, HepG2 cells, 293T cells, and 293F suspension cells.

Functionalized PDA/DEX-PEI@HA nanoparticles combined with sleeping-beauty transposons for multistage targeted delivery of CRISPR/Cas9 gene.

CRISPR/Cas9 system has been used as the most powerful gene editing tool for precision medicine and advanced gene therapy. However, its wide applications are limited by the poor biosafety of lentivirus delivery vectors though with high-efficiency transduction. To construct a safer vector and promote genome integration, the CRISPR/Cas9 gene is cloned into a plasmid-based non-viral safe vector Sleeping-Beauty (SB) transposon in this study to obtain pT2SpCas9. Meanwhile, PDA/DEX-PEI@HA (PDPH) nanoparticles are constructed to facilitate the precise CRISPR/Cas9 targeting delivery, by using polydopamine (PDA) as the carrier, hyaluronic acid (HA) as the cell-targeting ligand and dexamethasone (DEX) as the nuclear localization signal (NLS). The results showed that PDPH could deliver pDNA efficiently into the cell and further into the nucleus. The transfection efficiency of PDPH is much higher than that of NPs without HA and DEX. Remarkably, the cytotoxicity of PDPH is negligible in comparison to PEI25k and PEI10k. Western blots showed that after the transfection of PDPH/pT2SpCas9-Nanog/SB11, Nanog protein in HeLa cells is knocked out, and the proliferation and migration abilities of tumor cells are significantly decreased. This study demonstrates that PDA/DEX-PEI25k@HA/pT2SpCas9 (PDPH25 K/pT2SpCas9) has the great potential as a non-viral gene vector for CRISPR/Cas9 delivery and clinical medication.

Degradable cationic polyesters via ring-opening copolymerization of valerolactones as nanocarriers for the gene delivery.

The development of cationic polymers as non-viral gene vectors has been hurdled by their high toxicity, thus degradable and biocompatible polymers are urgently demanded. Herein, five polyesters (B3a-B3e) were synthesized based on the ring-opening copolymerization between α-allyl-δ-valerolactone and δ-valerolactone derivatives decorated with alkyl or alkoxyl chains of different lengths, followed by the modification with 1,5,9-triazacyclododecyl ([12]aneN3) through thiol-ene click reactions. The five polyesters effectively condensed DNA into nanoparticles. Of them, B3a with a shorter alkyl chain and B3d with more positive charged units showed stronger DNA condensing performance and can completely retard the migration of DNA at N/P = 1.6 in the presence of DOPE. B3b/DOPE with a longer alkyl chain exhibited the highest transfection efficiency in HeLa cells with 1.8 times of 25 kDa PEI, while B3d/DOPE with more positive charged units exhibited highest transfection efficiency in A549 cells with 2.3 times of 25 kDa PEI. B3b/DOPE and B3d/DOPE successfully delivered pEGFP into zebrafish, which was superior to 25 kDa PEI (1.5 folds and 1.1 folds, respectively). The cytotoxicity measurements proved that the biocompatibility of these polyesters was better than 25 kDa PEI, due to their degradable property in acid environment. The results indicated that these cationic polyesters can be developed as potential non-viral gene vectors for DNA delivery.

Quantitation of Trace Levels of DNA Released from Disrupted Adeno-Associated Virus Gene Therapy Vectors.

Adeno-associated virus (AAV) vectors for gene therapy have potential to provide a durable treatment response for a number of diseases with unmet need. DNA is released from AAV capsids at high temperatures. Less is known about DNA release that may occur under conditions relevant to clinical and commercial manufacturing, storage, and distribution. In this work we developed and applied a sensitive fluorescent dye-based method to quantitate trace levels of DNA released from AAV capsids. The method was used to characterize the impact of manufacturing process steps on the increase (up to 1.5%) and removal (down to 0.2%) of free DNA. Free DNA increased by 0.3% per day at 37 °C and by 0.4% per freeze/thaw cycle in a phosphate-buffered saline formulation. When stored for 2 years at different temperatures, free DNA remained low (<0.6%) at both ≤ -60 °C and at 2-8 °C but was higher (2.6%) when the same sample was stored at -20 °C. The dye-based method may be used to further characterize release of free DNA for different processes, formulations, and stress conditions. Overall, release of free DNA was a relatively minor degradation pathway under the conditions studied in this work.