Impact of Formulation Conditions on Lipid Nanoparticle Characteristics and Functional Delivery of CRISPR RNP for Gene Knock-Out and Correction

The CRISPR-Cas9 system is an emerging therapeutic tool with the potential to correct diverse ge-netic disorders. However, for gene therapy applications an efficient delivery vehicle is required, capable of delivering the CRISPR-Cas9 components into the cytosol of the intended target cell population. Once there, the ribonucleoprotein complex (RNP) can be transported into the nucleus. Lipid nanoparticles (LNP) serve as promising candidates for delivery of CRISPR-Cas9 RNP. These delivery vehicles have been optimized for the delivery of nucleic acids, such as mRNA. Co-delivery of Cas9 encoding mRNA with the accompanying sgRNA leads to translation of the Cas9 protein and formation of the Cas9 RNP inside the cell. Only recently, direct delivery of the CRISPR-Cas9 RNP complexes has been explored, which requires adjustments to the LNP formulation. In this study, the importance of buffer composition and cationic charge during RNP and ssDNA en-trapment in LNP are demonstrated. After optimizing several formulation parameters, LNP were prepared that were colloidally stable in human plasma and efficiently deliver the SpCas9 RNP and ssDNA for HDR-correction in reporter cells. Under optimal formulation conditions, gene knock-out and gene correction efficiencies as high as 80% and 20%, respectively were achieved at nanomolar CRISPR-Cas9 RNP concentrations.

NgCAM and VAMP2 Reveal that Direct Delivery and Dendritic Degradation Maintain Axonal Polarity

Neurons are polarized cells of extreme scale and compartmentalization. To fulfill their role in electrochemical signaling, axons must maintain a specific complement of membrane proteins. Despite being subject of considerable attention, the trafficking pathway of axonal membrane proteins is not well understood. Two pathways, direct delivery and transcytosis, have been proposed. Previous studies reached contradictory conclusions about which of these mediates delivery of axonal membrane proteins to their destination, in part because they evaluated long-term distribution changes and not vesicle transport. We developed a novel strategy to selectively label vesicles in different trafficking pathways and determined the trafficking of two canonical axonal membrane proteins, NgCAM and VAMP2. Results from detailed quantitative analyses of transporting vesicles differed substantially from previous studies and found that axonal membrane proteins overwhelmingly undergo direct delivery. Transcytosis plays only a minor role in axonal delivery of these proteins. In addition, we identified a novel pathway by which wayward axonal proteins that reach the dendritic plasma membrane are targeted to lysosomes. These results redefine how axonal proteins achieve their polarized distribution, a crucial requirement for elucidating the underlying molecular mechanisms. [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Streptomycin Sulfate–Loaded Niosomes Enables Increased Antimicrobial and Anti-Biofilm Activities

One of the antibiotics used to treat infections is streptomycin sulfate that inhibits both Gram-negative and -positive bacteria. Nanoparticles are suitable carriers for the direct delivery and release of drug agents to infected locations. Niosomes are one of the new drug delivery systems that have received much attention today due to their excellent biofilm penetration property and controlled release. In this study, niosomes containing streptomycin sulfate were prepared by using the thin layer hydration method and optimized based on the size, polydispersity index (PDI), and encapsulation efficiency (EE%) characteristics. It was found that the Span 60-to-Tween 60 ratio of 1.5 and the surfactant-to-cholesterol ratio of 1.02 led to an optimum formulation with a minimum of size, low PDI, and maximum of EE of 97.8 nm, 0.27, and 86.7%, respectively. The drug release investigation showed that 50.0 ± 1.2% of streptomycin sulfate was released from the niosome in 24 h and reached 66.4 ± 1.3% by the end of 72 h. Two-month stability studies at 25° and 4°C showed more acceptable stability of samples kept at 4°C. Consequently, antimicrobial and anti-biofilm activities of streptomycin sulfate–loaded niosomes against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were found significantly higher than those of free drug, and the minimum inhibitory concentration values decreased 4- to 8-fold. Furthermore, niosome-encapsulated streptomycin up to 1,500 μg/ml exhibited negligible cytotoxicity against the human foreskin fibroblasts cell line, whereas the free drug exhibited slight cytotoxicity at this concentration. Desired physical characteristics and low toxicity of niosomal nano-carriers containing streptomycin sulfate made them a demanded candidate for the treatment of current bacterial infections and biofilms.

CRISPR/Cas9 System-Mediated Gene Editing in the Fujian Oysters (Crassostrea angulate) by Electroporation

The Fujian oyster (Crassostrea angulate) is an important marine bivalve mollusk with high economic value. Gene function research and gene editing techniques have broad application prospects in oyster. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for genome engineering in many species. CRISPR-mediated gene editing has also been used successfully in the Pacific oyster through direct delivery of the CRISPR/Cas9 components into oyster embryos by microinjection. However, the low throughput and operational difficulties associated with microinjection is one of the factors limiting the widespread application of CRISPR/Cas9 in oysters. In this study, we attempted to deliver the CRISPR/Cas9-system into the embryos of C. angulate by electroporation. An all-in-one CRISPR/Cas9 vector plasmid was used as CRISPR/Cas9 system in this study. Electroporation was carried out using both eggs and blastula larvae. A large number of larvae became malformed or die after electroporation. A single base substitution mutation was detected in the D-larvae developed from electroporated eggs. Our results demonstrate that the CRISPR/Cas9 system can be delivered into embryos of C. angulate for gene editing by electroporation, which provides a reference and will further contribute to the future application of electroporation in mollusks.

Direct Delivery of ANA-TA9, a Peptide Capable of Aβ Hydrolysis, to the Brain by Intranasal Administration

We have recently reported Catalytides (Catalytic peptides) JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMI), which are the first Catalytides found to cleave Aβ42. Although the Catalytides must be delivered to the brain parenchyma to treat Alzheimer’s disease, the blood–brain barrier (BBB) limits their entry into the brain from the systemic circulation. To avoid the BBB, the direct route from the nasal cavity to the brain was used in this study. The animal studies using rats and mice clarified that the plasma clearance of ANA-TA9 was more rapid than in vitro degradation in the plasma, whole blood, and the cerebrospinal fluid (CSF). The brain concentrations of ANA-TA9 were higher after nasal administration than those after intraperitoneal administration, despite a much lower plasma concentration after nasal administration, suggesting the direct delivery of ANA-TA9 to the brain from the nasal cavity. Similar findings were observed for its transport to CSF after nasal and intravenous administration. The concentration of ANA-TA9 in the olfactory bulb reached the peak at 5 min, whereas those in the frontal and occipital brains was 30 min, suggesting the sequential backward translocation of ANA-TA9 in the brain. In conclusion, ANA-TA9 was efficiently delivered to the brain by nasal application, as compared to other routes.

Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants

Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome editing (GE). The predominant feature of agroinfiltration is the abolishment of Transfer-DNA (T-DNA) integration event to ensure fewer biosafety and regulatory issues besides showcasing the capability to perform transcription and translation efficiently, hence providing a large picture through pilot-scale experiment via transient approach. The direct delivery of recombinant agrobacteria through this approach carrying CRISPR/Cas cassette to knockout the expression of the target gene in the intercellular tissue spaces by physical or vacuum infiltration can simplify the targeted site modification. This review aims to provide information on Agrobacterium-mediated transformation and implementation of agroinfiltration with GE to widen the horizon of targeted genome editing before a stable genome editing approach. This will ease the screening of numerous functions of genes in different plant species with wider applicability in future.

Metal and Metal Oxide Nanoparticle as a Novel Antibiotic Carrier for the Direct Delivery of Antibiotics

In addition to the benefits, increasing the constant need for antibiotics has resulted in the development of antibiotic bacterial resistance over time. Antibiotic tolerance mainly evolves in these bacteria through efflux pumps and biofilms. Leading to its modern and profitable uses, emerging nanotechnology is a significant field of research that is considered as the most important scientific breakthrough in recent years. Metal nanoparticles as nanocarriers are currently attracting a lot of interest from scientists, because of their wide range of applications and higher compatibility with bioactive components. As a consequence of their ability to inhibit the growth of bacteria, nanoparticles have been shown to have significant antibacterial, antifungal, antiviral, and antiparasitic efficacy in the battle against antibiotic resistance in microorganisms. As a result, this study covers bacterial tolerance to antibiotics, the antibacterial properties of various metal nanoparticles, their mechanisms, and the use of various metal and metal oxide nanoparticles as novel antibiotic carriers for direct antibiotic delivery.

Cellular Therapy for the Treatment of Paediatric Respiratory Disease

Respiratory disease is the leading cause of death in children under the age of 5 years old. Currently available treatments for paediatric respiratory diseases including bronchopulmonary dysplasia, asthma, cystic fibrosis and interstitial lung disease may ameliorate symptoms but do not offer a cure. Cellular therapy may offer a potential cure for these diseases, preventing disease progression into adulthood. Induced pluripotent stem cells, mesenchymal stromal cells and their secretome have shown great potential in preclinical models of lung disease, targeting the major pathological features of the disease. Current research and clinical trials are focused on the adult population. For cellular therapies to progress from preclinical studies to use in the clinic, optimal cell type dosage and delivery methods need to be established and confirmed. Direct delivery of these therapies to the lung as aerosols would allow for lower doses with a higher target efficiency whilst avoiding potential effect of systemic delivery. There is a clear need for research to progress into the clinic for the treatment of paediatric respiratory disease. Whilst research in the adult population forms a basis for the paediatric population, varying disease pathology and anatomical differences in paediatric patients means a paediatric-centric approach must be taken.

PENTINGNYA PENGATURAN DIET HIPERTENSI DALAM PENCEGAHAN KEKAMBUHAN HIPERTENSI

Hipertensi adalah salah satu penyakit terbanyak yang dijumpai pada lansia, dan secara khusus di Kampung KB dan Paya Lebar Kelurahan Tanjung Uban Kota Hipertensi juga merupakan penyakit yang mendominasi di kalangan usia dewasa akhir dan lansia. Seseorang dikatakan menderita hipertensi apabila memiliki tekanan darah sistolik lebih dari 140 mmHg dan diastolik lebih dari 90 mmHg. Berdasarkan penelusuran awal diketahui sebanyak 60% penderita hipertensi di Kampung KB dan Paya Lebar memiliki tekanan darah yang tidak terkontrol Kendala yang sering dialami oleh Lansia dalam mengontrol tekanan darah yaitu 60% mengatakan makanan yang sulit tanpa garam, 20% mengatakan tidak ada yang mengantar ke puskesmas sehingga kadang putus obat dan 20% mengatakan bosan dengan mengkomsumsi obat terus menerus. Tujuan dari penyuluhan ini adalah untuk memberikan pengetahuan tentang diet hipertensi agar peserta dapat melakukan pencegahan dan perawatan pada penderita hipertensi dengan benar dengan bahan yang mdah didapatkan. Metode yang dilakukan adalah “direct delivery” atau penyampaian secara langsung dengan tetap protocol kesehatan Covid-19. Peserta berjumlah 15 orang yang terdiri dari lansia dan kader daerah Kampung KB dan Paya Lebar Kelurahan Tanjung Uban Kota. Hasil dari penyuluhan ini adalah bertambahnya ilmu pengetahuan para peserta mengenai pencegahan dan perawatan pada penderita hipertensi yang ditandai dengan 15 peserta (100%) dapat menjawab pertanyaan tentang diet hipertensi. Kesimpulannya adalah pengetahuan peserta tentang diet Hipertensi menunjukkan peningkatan sangat baik.

PENTINGNYA PENGATURAN DIET HIPERTENSI DALAM PENCEGAHAN KEKAMBUHAN HIPERTENSI

Hipertensi adalah salah satu penyakit terbanyak yang dijumpai pada lansia, dan secara khusus di Kampung KB dan Paya Lebar Kelurahan Tanjung Uban Kota Hipertensi juga merupakan penyakit yang mendominasi di kalangan usia dewasa akhir dan lansia. Seseorang dikatakan menderita hipertensi apabila memiliki tekanan darah sistolik lebih dari 140 mmHg dan diastolik lebih dari 90 mmHg. Berdasarkan penelusuran awal diketahui sebanyak 60% penderita hipertensi di Kampung KB dan Paya Lebar memiliki tekanan darah yang tidak terkontrol Kendala yang sering dialami oleh Lansia dalam mengontrol tekanan darah yaitu 60% mengatakan makanan yang sulit tanpa garam, 20% mengatakan tidak ada yang mengantar ke puskesmas sehingga kadang putus obat dan 20% mengatakan bosan dengan mengkomsumsi obat terus menerus. Tujuan dari penyuluhan ini adalah untuk memberikan pengetahuan tentang diet hipertensi agar peserta dapat melakukan pencegahan dan perawatan pada penderita hipertensi dengan benar dengan bahan yang mdah didapatkan. Metode yang dilakukan adalah “direct delivery” atau penyampaian secara langsung dengan tetap protocol kesehatan Covid-19. Peserta berjumlah 15 orang yang terdiri dari lansia dan kader daerah Kampung KB dan Paya Lebar Kelurahan Tanjung Uban Kota. Hasil dari penyuluhan ini adalah bertambahnya ilmu pengetahuan para peserta mengenai pencegahan dan perawatan pada penderita hipertensi yang ditandai dengan 15 peserta (100%) dapat menjawab pertanyaan tentang diet hipertensi. Kesimpulannya adalah pengetahuan peserta tentang diet Hipertensi menunjukkan peningkatan sangat baik.