Spermine-Based Poly(ß-amino ester)s for siRNA Delivery against Mutated KRAS in Lung Cancer.

Polyethylenimine (PEI) is a highly efficient cationic polymer for nucleic acid delivery, and although it is commonly used in preclinical studies, its clinical application is limited because of concerns regarding its cytotoxicity. Poly(ß-amino ester)s are a new group of biodegradable and biocompatible cationic polymers that can be used for siRNA delivery. In this study, we synthesized Boc-protected and deprotected poly(ß-amino ester)s, P(BSpBAE) and P(SpBAE), respectively, based on spermine and 1,4-butanediol diacrylate to deliver siRNA. The polymers were synthesized by Michael addition in a step-growth polymerization and characterized via 1H NMR spectroscopy and size-exclusion chromatography (SEC). The polymers can encapsulate siRNA as determined by SYBR gold assays. Both polymers and polyplexes were biocompatible in vitro. Furthermore, the cellular uptake of P(BSpBAE) and P(SpBAE) polyplexes was more efficient than for branched PEI (25 kDa) polyplexes at the same N/P ratios. P(BSpBAE) polyplexes achieved 60% eGFP knockdown in vitro, which indicates that the Boc-protection can improve the siRNA delivery and gene silencing efficiency of PBAEs. P(BSpBAE) polyplexes and P(SpBAE) polyplexes showed different cellular uptake mechanisms, and P(BSpBAE) polyplexes demonstrated decreased endosomal entrapment, which could explain why P(BSpBAE) polyplexes more efficiently mediated gene silencing than P(SpBAE) polyplexes. Furthermore, transfection of an siRNA against mutated KRAS in KRAS-mutated lung cancer cells led to around 35% (P(BspBAE)) to 45% (P(SpBAE)) inhibition of KRAS expression and around 33% (P(SpBAE)) to 55% (P(BspBAE)) decreased motility in a migration assay. These results suggest that the newly developed spermine-based poly(ß-amino ester)s are promising materials for therapeutic siRNA delivery.

ciRS-7 is a prognostic biomarker and potential gene therapy target for renal cell carcinoma.

Circular RNAs are a new class of non-coding RNAs that have been shown to play critical roles in the development and progression of renal cell carcinoma (RCC). However, little is known about the functional mechanisms and therapeutic role of ciRS-7 in RCC. A series of in vitro and in vivo experiments were performed to investigate the functional mechanism and therapeutic role of ciRS-7, such as real-time quantitative PCR, CCK-8, wound healing, transwell, colony formation, Edu, tumor xenograft and lung metastasis in NSG mice. RNA pull-down, dual luciferase reporter, fluorescence in situ hybridization (FISH) and rescue assays were used to determine the relationship between ciRS-7, miR-139-3p and TAGLN. In addition, we constructed PBAE/si-ciRS-7 nanocomplexes with PBAE material to evaluate the therapeutic effect of the nanocomplexes on tumor in vivo. ciRS-7 was highly expressed in RCC tumor tissues and cell lines, and high ciRS-7 expression correlated with tumor size, high Fuhrman grade and poor survival. Depletion of ciRS-7 significantly inhibited RCC cell proliferation, invasion, tumor growth and metastasis in vivo, while overexpression of ciRS-7 had the opposite effect. Mechanistically, ciRS-7 acts as a "ceRNA" for miR-139-3p to prevent TAGLN degradation and promoting RCC progression and metastasis via the PI3K/AKT signaling pathway. In addition, miR-139-3p mimics or inhibitor could reverse the altered malignant tumor behavior caused by ciRS-7 overexpression or silencing. Furthermore, the PBAE/siciRS-7 nanocomplexes could significantly inhibit RCC tumor progression and metastasis in vivo. ciRS-7 acts as a tumor promoter by regulating the miR-139-3p/TAGLN axis and activating the PI3K/AKT signaling pathway to promote RCC progression and metastasis. Drug development of PBAE/si-ciRS-7 nanocomplexes targeting ciRS-7 may represent a promising gene therapeutic strategy for RCC.

Nanoparticles Based on Poly (ß-Amino Ester) and HPV16-Targeting CRISPR/shRNA as Potential Drugs for HPV16-Related Cervical Malignancy.

Persistent high-risk HPV infection is the main cause of cervical cancer. The HPV oncogene E7 plays an important role in HPV carcinogenesis. Currently, HPV vaccines do not offer an effective treatment for women who already present with cervical disease, and recommended periodical cervical screenings are difficult to perform in countries and areas lacking medical resources. Our aim was to develop nanoparticles (NPs) based on poly (ß-amino ester) (PBAE) and HPV16 E7-targeting CRISPR/short hairpin RNA (shRNA) to reduce the levels of HPV16 E7 as a preliminary form of a drug to treat HPV infection and its related cervical malignancy. Our NPs showed low toxicity in cells and mouse organs. By reducing the expression of HPV16 E7, our NPs could inhibit the growth of cervical cancer cells and xenograft tumors in nude mice, and they could reverse the malignant cervical epithelium phenotype in HPV16 transgenic mice. The performance of NPs containing shRNA is better than that of NPs containing CRISPR. HPV-targeting NPs consisting of PBAE and CRISPR/shRNA could potentially be developed as drugs to treat HPV infection and HPV-related cervical malignancy.

Optimization of a Degradable Polymer-Lipid Nanoparticle for Potent Systemic Delivery of mRNA to the Lung Endothelium and Immune Cells.

mRNA therapeutics hold great potential for treating a variety of diseases through protein-replacement, immunomodulation, and gene editing. However, much like siRNA therapy the majority of progress in mRNA delivery has been confined to the liver. Previously, we demonstrated that poly(ß-amino esters), a class of degradable polymers, are capable of systemic mRNA delivery to the lungs in mice when formulated into nanoparticles with poly(ethylene glycol)-lipid conjugates. Using experimental design, a statistical approach to optimization that reduces experimental burden, we demonstrate herein that these degradable polymer-lipid nanoparticles can be optimized in terms of polymer synthesis and nanoparticle formulation to achieve a multiple order-of-magnitude increase in potency. Furthermore, using genetically engineered Cre reporter mice, we demonstrate that mRNA is functionally delivered to both the lung endothelium and pulmonary immune cells, expanding the potential utility of these nanoparticles.

Biodegradable STING agonist nanoparticles for enhanced cancer immunotherapy.

Therapeutic cancer vaccines require adjuvants leading to robust type I interferon and proinflammatory cytokine responses in the tumor microenvironment to induce an anti-tumor response. Cyclic dinucleotides (CDNs), a potent Stimulator of Interferon Receptor (STING) agonist, are currently in phase I trials. However, their efficacy may be limited to micromolar concentrations due to the cytosolic residence of STING in the ER membrane. Here we utilized biodegradable, poly(beta-amino ester) (PBAE) nanoparticles to deliver CDNs to the cytosol leading to robust immune response at >100-fold lower extracellular CDN concentrations in vitro. The leading CDN PBAE nanoparticle formulation induced a log-fold improvement in potency in treating established B16 melanoma tumors in vivo when combined with PD-1 blocking antibody in comparison to free CDN without nanoparticles. This nanoparticle-mediated cytosolic delivery method for STING agonists synergizes with checkpoint inhibitors and has strong potential for enhanced cancer immunotherapy.

Poly-beta-amino-esters nano-vehicles based drug delivery system for cartilage.

The efficient delivery of therapeutic molecules to the cartilage of joints is a major obstacle in developing useful therapeutic interventions; hence, a targeted drug delivery system for this tissue is critical. We have overcome the challenge by developing a system that employs electrostatic attraction between the negatively charged constituents of cartilage and a positively charged polymer, poly-beta amino esters (PBAEs). We have demonstrated cartilage uptake of dexamethasone (DEX) covalently bound to the PBAE was doubled and retention in tissues prolonged compared to the equivalent dose of the commercial drug formulation. Moreover, no adverse effects on chondrocytes were found. Our data also show that PBAEs can bind not only healthy cartilage tissues but also enzymatically treated cartilage mimicking early stages of OA. Our PBAEs-prodrug technology's advantages are fourfold; the specificity and efficacy of its targeting mechanism for cartilage, the ease of its production and the low-cost nature of the delivery system.

In Situ Reprogramming of Immune Cells Using Synthetic Nanomaterials.

In the past decade, adoptive cell therapy with chimeric antigen receptor-T (CAR-T) cells has revolutionized cancer treatment. However, the complexity and high costs involved in manufacturing current adoptive cell therapy greatly inhibit its widespread availability and access. To address this, in situ cell therapy, which directly reprograms immune cells inside the body, has recently been developed as a promising alternative. Here, an overview of the recent progress in the development of synthetic nanomaterials is provided to deliver plasmid DNA or mRNA for in situ reprogramming of T cells and macrophages, focusing especially on in situ CAR therapies. Also, the main challenges for in situ immune cell reprogramming are discussed and some approaches to overcome these barriers to fulfill the clinical applications are proposed.

pH-Responsive Block Copolymer Micelles of Temsirolimus: Preparation, Characterization and Antitumor Activity Evaluation.

Purpose: Renal cell carcinoma (RCC) is the most common and lethal type of urogenital cancer, with one-third of new cases presenting as metastatic RCC (mRCC), which, being the seventh most common cancer in men and the ninth in women, poses a significant challenge. For patients with poor prognosis, temsirolimus (TEM) has been approved for first-line therapy, possessing pharmacodynamic activities that block cancer cell growth and inhibit proliferation-associated proteins. However, TEM suffers from poor water solubility, low bioavailability, and systemic side effects. This study aims to develop a novel drug formulation for the treatment of RCC. Methods: In this study, amphiphilic block copolymer (poly(ethylene glycol) monomethyl ether-poly(beta-amino ester)) (mPEG-PBAE) was utilized as a drug delivery vehicle and TEM-loaded micelles were prepared by thin-film hydration method by loading TEM inside the nanoparticles. Then, the molecular weight of mPEG-PBAE was controlled to make it realize hydrophobic-hydrophilic transition in the corresponding pH range thereby constructing pH-responsive TEM-loaded micelles. Characterization of pH-responsive TEM-loaded nanomicelles particle size, potential and micromorphology while its determination of drug-loading properties, in vitro release properties. Finally, pharmacodynamics and hepatorenal toxicity were further evaluated. Results: TEM loading in mPEG-PBAE increased the solubility of TEM in water from 2.6 µg/mL to more than 5 mg/mL. The pH-responsive TEM-loaded nanomicelles were in the form of spheres or spheroidal shapes with an average particle size of 43.83 nm and a Zeta potential of 1.79 mV. The entrapment efficiency (EE) of pH-responsive TEM nanomicelles with 12.5% drug loading reached 95.27%. Under the environment of pH 6.7, the TEM was released rapidly within 12 h, and the release rate could reach 73.12% with significant pH-dependent characteristics. In vitro experiments showed that mPEG-PBAE preparation of TEM-loaded micelles had non-hemolytic properties and had significant inhibitory effects on cancer cells. In vivo experiments demonstrated that pH-responsive TEM-loaded micelles had excellent antitumor effects with significantly reduced liver and kidney toxicity. Conclusion: In conclusion, we successfully prepared pH-responsive TEM-loaded micelles. The results showed that pH-responsive TEM-loaded micelles can achieve passive tumor targeting of TEM, and take advantage of the acidic conditions in tumor tissues to achieve rapid drug release.

Gastrointestinal Delivery of an mRNA Vaccine Using Immunostimulatory Polymeric Nanoparticles.

mRNA vaccines can be translated into protein antigens, in vivo, to effectively induce humoral and cellular immunity against these proteins. While current mRNA vaccines have generated potent immune responses, the need for ultracold storage conditions (- 80 °C) and healthcare professionals to administer the vaccine through the parenteral route has somewhat limited their distribution in rural areas and developing countries. Overcoming these challenges stands to transform future deployment of mRNA vaccines. In this study, we developed an mRNA vaccine that can trigger a systemic immune response through administration via the gastrointestinal (GI) tract and is stable at 4 °C. A library of cationic branched poly(ß-amino ester) (PBAE) polymers was synthesized and characterized, from which a polymer with high intracellular mRNA delivery efficiency and immune stimulation capacity was down-selected. mRNA vaccines made with the lead polymer-elicited cellular and humoral immunity in mice. Furthermore, lyophilization conditions of the formulation were optimized to enable storage under refrigeration. Our results suggest that PBAE nanoparticles are potent mRNA delivery platforms that can elicit B cell and T cell activation, including antigen-specific cellular and humoral responses. This system can serve as an easily administrable, potent oral mRNA vaccine.

Nanocomposite orthopaedic bone cement combining long-acting dual antimicrobial drugs.

Antibiotic loaded bone cements are widely used in total joint replacement (TJR); despite many limitations such as a burst release which leads to antibiotic concentration below inhibitory levels and possibly contributing to the selection of antibiotic resistant strains. In order to address such limitations and to simultaneously address antibiotic resistance and short-term antimicrobial activity, we developed a nanocomposite bone cement capable of providing a controlled release of antimicrobial agents from bone cement to act as prophylaxis or treatment against prosthetic joint infections (PJIs). Gentamicin and chlorhexidine were loaded in combination on silica nanoparticles surface using layer-by-layer coating technique (LbL) combining hydrolysable and non-hydrolysable polymers. The drug release from the nanocomposite continued for >50 days at concentrations higher than the commercial formulation containing the same amount of antimicrobial drugs, where burst release for few days were observed. Moreover, the nanocomposite bone cement showed superior antimicrobial inhibition without adversely affecting the mechanical properties or the ability of osteoblasts to grow. In vivo experiments with an infected bone lesion model along with mass-spectrometric analysis also provided further evidence of efficacy and safety of the implanted nanocomposite material as well as its prolonged drug eluting profile. The developed nanocomposite bone cement has the potential to reduce PJIs and enable treatment of resistant established infections; moreover, the newly developed LbL based nano-delivery system may also have wider applications in reducing the threat posed by antimicrobial resistance.

pH-sensitive nano-polyelectrolyte complexes with arthritic macrophage-targeting delivery of triptolide.

Since pro-inflammatory macrophages take on a critical significance in the pathophysiology of rheumatoid arthritis (RA), the therapeutics to affect macrophages may receive distinct anti-RA effects. However, the therapeutic outcomes are still significantly impeded, which is primarily due to the insufficient drug delivery at the arthritic site. In this study, the macrophage-targeting and pH stimuli-responsive nano-polyelectrolyte complexes were designed for the efficient targeted delivery of triptolide (TP/PNPs) on the arthritic site. The anionic and cationic amphiphilic copolymers, i.e., hyaluronic acid-g-vitamin E succinate (HA-VE) and the quaternized poly (ß-amino ester) (QPBAE-C18), were prepared and then characterized. The result indicated that TP/PNPs with the uniform particle size of âˆ¼ 175 nm exhibited the high drug loading capacity and storage stability based on the polymeric charge interaction, in which DLC and DEE of TP/PNPs were obtained as 11.27 ± 0.44 % and 95.23 ± 2.34 %, respectively. Mediated by the "ELVIS" effect of NPs, CD44 receptor-mediated macrophage targeting, and pH-sensitive endo/lysosomal escape under the "proton sponge" effect, TP/PNPs exhibited the enhanced cellular internalization and cytotoxicity while mitigating the inflammation of LPS-activated RAW 264.7 cells. Even after 96-hour after administration, PNPs were preferentially accumulated in the inflammatory joints in a long term. It is noteworthy that after treatment for 14 days with 100 µg/kg of TP, TP/PNPs significantly facilitated arthritic symptom remission, protected cartilage, and mitigated inflammation of antigen-induced arthritis (AIA) rats, whereas the systematic side-effects of TP were reduced. In this study, an effective drug delivery strategy was proposed for the treatment of RA.

Corrigendum: Treatment of surgical brain injury by immune tolerance induced by peripheral intravenous injection of biotargeting nanoparticles loaded with brain antigens.

[This corrects the article DOI: 10.3389/fimmu.2019.00743.].

Synthesis and Electrochemical Evaluation of MSNs-PbAE Nanocontainers for the Controlled Release of Caffeine as a Corrosion Inhibitor.

In this paper, a controlled-release system of caffeine as a corrosion inhibitor was obtained by encapsulating it in MCM-41 silica nanoparticles coated with a poly(ß-amino ester) (PbAE), a pH-sensible polymer. Encapsulation was verified using Fourier transform infrared spectroscopy (FTIR) and thermogravimetry (TGA). The release of caffeine from the nanocontainers was analyzed in electrolytes with pH values of 4, 5, and 7 using UV-Vis, showing a 21% higher release in acidic electrolytes than in neutral electrolytes, corroborating its pH sensitivity. Electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization were used to determine the inhibition mode and efficiency of the encapsulated and free caffeine. The caffeine released from the nanocontainers showed the highest efficiency, which was 85.19%. These results indicate that these nanocontainers could have potential use in smart anticorrosion coating applications.

A design approach for layer-by-layer surface-mediated siRNA delivery.

The ability to coat scaffolds and wound dressings with therapeutic short interfering RNA (siRNA) holds much potential for applications in wound healing, cancer treatment, and regenerative medicine. Layer-by-layer (LbL) technology is an effective method to formulate polyelectrolyte thin films for local delivery of siRNA; however, the formation and efficacy of LbL coatings as drug delivery systems are highly contingent on the assembly conditions. Here, we investigate the effects of LbL assembly parameters on film composition and consequent siRNA-mediated gene knockdown efficiency in vitro. Films comprising poly(ß-amino ester) (PBAE) and siRNA were built on polyglactin 910 (Vicryl) sutures consisting of poly(10% L-lactide, 90% glycolide). A fractional factorial design was employed, varying the following LbL assembly conditions: pH, ionic strength, PBAE concentration, and siRNA concentration. Effects of these parameters on PBAE loading, siRNA loading, their respective weight ratios, and in vitro siRNA-mediated knockdown were elucidated. The parameter effects were leveraged to create a rationally designed set of solution conditions that was predicted to give effective siRNA-mediated knockdown, but not included in any of the original experimental conditions. This level of knockdown with our rationally designed loading conditions (47%) is comparable to previous formulations from our lab while being simpler in construction and requiring fewer film layers, which could save time and cost in manufacturing. This study highlights the importance of LbL solution conditions in the preparation of surface-mediated siRNA delivery systems and presents an adaptable methodology for extending these electrostatically-assembled coatings to the delivery of other therapeutic nucleic acids. STATEMENT OF SIGNIFICANCE: Short interfering RNA (siRNA) therapeutics are powerful tools to silence aberrant gene expression in the diseased state; however, the clinical utility of these therapies relies on effective controlled delivery approaches. Electrostatic self-assembly through the layer-by-layer (LbL) process enables direct siRNA release from surfaces, but this method is highly dependent upon the specific solution conditions used. Here, we use a fractional factorial design to illustrate how these assembly conditions impact composition of siRNA-eluting LbL thin films. We then elucidate how these properties mediate in vitro transfection efficacy. Ultimately, this work presents a significant step towards understanding how optimization of assembly conditions for surface-mediated LbL delivery can promote transfection efficacy while reducing the processing and material required.

E7-Targeted Nanotherapeutics for Key HPV Afflicted Cervical Lesions by Employing CRISPR/Cas9 and Poly (Beta-Amino Ester).

INTRODUCTION: Persistent HR-HPV (high-risk human papillomavirus) infection is the main cause of cervical cancer. The HPV oncogene E7 plays a key role in HPV tumorigenesis. At present, HPV preventive vaccines are not effective for patients who already have a cervical disease, and implementation of the recommended regular cervical screening is difficult in countries and regions lacking medical resources. Therefore, patients need medications to treat existing HPV infections and thus block the progression of cervical disease. METHODS: In this study, we developed nanoparticles (NPs) composed of the non-viral vector PBAE546 and a CRISPR/Cas9 recombinant plasmid targeting HPV16 E7 as a vaginal treatment for HPV infection and related cervical malignancies. RESULTS: Our NPs showed low toxicity and high biological safety both in vitro (cell line viability) and in vivo (various important organs of mice). Our NPs significantly inhibited the growth of xenograft tumors derived from cervical cancer cell lines in nude mice and significantly reversed the cervical epithelial malignant phenotype of HPV16 transgenic mice. CONCLUSION: Our NPs have great potential to be developed as a drug for the treatment of HPV-related cervical cancer and precancerous lesions.

Poly(beta-amino ester) nanoparticles enable tumor-specific TRAIL secretion and a bystander effect to treat liver cancer.

Despite initial promise, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-based approaches to cancer treatment have yet to yield a clinically approved therapy, due to delivery challenges, a lack of potency, and drug resistance. To address these challenges, we have developed poly(beta-amino ester) (PBAE) nanoparticles (NPs), as well as an engineered cDNA sequence encoding a secretable TRAIL (sTRAIL) protein, to enable reprogramming of liver cancer cells to locally secrete TRAIL protein. We show that sTRAIL initiates apoptosis in transfected cells and has a bystander effect to non-transfected cells. To address TRAIL resistance, NP treatment is combined with histone deacetylase inhibitors, resulting in >80% TRAIL-mediated cell death in target cancer cells and significantly slowed xenograft tumor growth. This anti-cancer effect is specific to liver cancer cells, with up to 40-fold higher cell death in HepG2 cancer cells over human hepatocytes. By combining cancer-specific TRAIL NPs with small-molecule-sensitizing drugs, this strategy addresses multiple challenges associated with TRAIL therapy and offers a new potential approach for cancer treatment.

Electrostatic Coating of Viral Particles for Gene Delivery Applications in Muscular Dystrophies: Influence of Size on Stability and Antibody Protection.

BACKGROUND: Duchenne Muscular Dystrophy (DMD) is one of the most common muscular dystrophies, caused by mutated forms of the dystrophin gene. Currently, the only treatment available is symptoms management. Novel approximations are trying to treat these patients with gene therapy, namely, using viral vectors. However, these vectors can be recognized by the immune system decreasing their therapeutic activity and making impossible a multidose treatment due to the induction of the humoral immunity following the first dose. OBJECTIVE: Our objective is to demonstrate the feasibility of using a hybrid vector to avoid immune clearance, based on the electrostatic coating of adeno-associated virus (AAVs) vectors with our proprietary polymers. METHODS: We coated model adeno-associated virus vectors by electrostatic interaction of our cationic poly (beta aminoester) polymers with the viral anionic capsid and characterized biophysical properties. Once the nanoformulations were designed, we studied their in vivo biodistribution by bioluminescence analysis and we finally studied the capacity of the polymers as potential coatings to avoid antibody neutralization. RESULTS: We tested two polymer combinations and we demonstrated the need for poly(ethylene glycol) addition to avoid vector aggregation after coating. In vivo biodistribution studies demonstrated that viral particles are located in the liver (short times) and also in muscles (long times), the target organ. However, we did not achieve complete antibody neutralization shielding using this electrostatic coating. CONCLUSIONS: The null hypothesis stands: although it is feasible to coat viral particles by electrostatic interaction with a proprietary polymer, this strategy is not appropriate for AAVs due to their small size, so other alternatives are required as a novel treatment for DMD patients.

Biodegradable Hydrogel Scaffolds Based on 2-Hydroxyethyl Methacrylate, Gelatin, Poly(ß-amino esters), and Hydroxyapatite.

New composite 3D scaffolds were developed as a combination of synthetic polymer, poly(2-hydroxyethyl methacrylate) (PHEMA), and a natural polymer, gelatin, with a ceramic component, nanohydroxyapatite (ID nHAp) dopped with metal ions. The combination of a synthetic polymer, to be able to tune the structure and the physicochemical and mechanical properties, and a natural polymer, to ensure the specific biological functions of the scaffold, with inorganic filler was applied. The goal was to make a new material with superior properties for applications in the biomedical field which mimics as closely as possible the native bone extracellular matrix (ECM). Biodegradable PHEMA hydrogel was obtained by crosslinking HEMA by poly(ß-amino esters) (PBAE). The scaffold's physicochemical and mechanical properties, in vitro degradation, and biological activity were assessed so to study the effects of the incorporation of nHAp in the (PHEMA/PBAE/gelatin) hydrogel, as well as the effect of the different pore-forming methods. Cryogels had higher elasticity, swelling, porosity, and percent of mass loss during degradation than the samples obtained by porogenation. The composite scaffolds had a higher mechanical strength, 10.14 MPa for the porogenated samples and 5.87 MPa for the cryogels, but a slightly lower degree of swelling, percent of mass loss, and porosity than the hybrid ones. All the scaffolds were nontoxic and had a high cell adhesion rate, which was 15-20% higher in the composite samples. Cell metabolic activity after 2 and 7 days of culture was higher in the composites, although not statistically different. After 28 days, cell metabolic activity was similar in all scaffolds and the TCP control. No effect of integrating nHAp into the scaffolds on osteogenic cell differentiation could be observed. Synergetic effects occurred which influenced the mechanical behavior, structure, physicochemical properties, and interactions with biological species.

Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion.

The CRISPR-Cas9 system is a powerful gene-editing tool with wide-ranging applications, but the safe and efficient intracellular delivery of CRISPR components remains a challenge. In this study, we utilized biodegradable poly(beta-amino ester) nanoparticles to codeliver plasmid DNA encoding Cas9 and short guide RNA (sgRNA), respectively, to enable gene knockout following a CRISPR-mediated cleavage at one genomic site (1-cut edit), as well as gene deletion following DNA cleavage at two sites flanking a region of interest (2-cut edits). We designed a reporter system that allows for easy evaluation of both types of edits: gene knockout can be assessed by a decrease in near-infrared fluorescent protein (iRFP) fluorescence, whereas deletion of an expression stop cassette turns on a red-enhanced nanolantern fluorescence/luminescence dual reporter. Nanoparticles enabled up to 70% gene knockout due to small indels, as well as 45% gain-of-function expression after a 600-bp deletion edit. The efficiency of 2-cut edits is more sensitive than 1-cut edits to Cas9 and the sgRNA expression level. We demonstrate promising biodegradable nanoparticle formulations for gene editing. Our findings also provide new insights into the screening and transfection requirements for different types of gene edits, which are applicable for designing nonviral delivery systems for the CRISPR-Cas9 platform.

Development of a Gene Delivery System Composed of a Cell-Penetrating Peptide and a Nontoxic Polymer.

Major concerns have arisen with respect to using viral vectors for gene therapies. Collateral effects include cancer resistance, development of new cancers, and even systemic deaths. For this reason, researchers have focused on the alternative of using nonviral nanocarriers for gene therapy. In this study, a gene delivery nanocarrier was developed, comprising a cell-penetrating peptide called WTAS as a primary nanocarrier and a poly(ß-amino ester) (PBAE) polymer as a secondary nanocarrier. Here, the PBAE polymer is used to protect the WTAS peptide from early degradation while further facilitating the transportation into cells. WTAS is a peptide that penetrates cell nuclei within a few minutes after exposure, which makes it an ideal candidate to transport genetic materials. The PBAE-WTAS nanocarrier was assembled and tested against three cell lines (NSC, B16F10, and GL26). Cytotoxic studies demonstrated the relatively low toxicity of the PBAE-WTAS nanocarrier and PBAE-WTAS loaded with green fluorescent protein (GFP) plasmid DNA (pDNA@PBAE-WTAS) against all three cell lines. Cell transfection experiments were carried out using GL26 cells. These studies demonstrated a very high transfection rate of PBAE-WTAS loaded with GFP plasmid DNA, leading to virtually complete transfection (> 90%). In conclusion, we report a very promising gene delivery nanocarrier, which can be further modified to transport a variety of genetic materials for targeted therapy of multiple diseases.