Drug administration via hollow microneedles (HMN) have the advantages of painlessness, avoidance of first-pass effect, capability of sustained infusion, and no need for professional personnel operation. In addition, HMN can also be applied in the fields of body fluid extraction and biosensors, showing broad application prospects. However, traditional manufacturing technologies cannot meet the demand for low-cost mass production of HMN, limiting its widespread application. This paper reviews the main manufacturing technologies used for HMN in recent years, which include photolithography and etching, laser etching, sputtering and electroplating, micro-molding, three-dimensional (3D) printing and drawing lithography. It further analyzes the characteristics and limitations of existing manufacturing technologies and points out that the combination of various manufacturing technologies can improve production efficiency to a certain extent. In addition, this paper looks forward to the future trends of HMN manufacturing technology and proposes possible directions for its development. In conclusion, it is expected that this review can provide new ideas and references for follow-up research.
Printing, Three-Dimensional ; Needles ; Humans ; Drug Delivery Systems/methods* ; Equipment Design ; Microinjections/methods*

Compared with conventional transdermal drug delivery systems, dissolving microneedles significantly enhance drug bioavailability by penetrating the stratum corneum barrier and achieving intradermal drug delivery. In order to improve the transdermal bioavailability of dexmedetomidine hydrochloride, in this study, a novel microneedle delivery system was developed for dexmedetomidine hydrochloride based on 3D printing combined with micro-molding. By systematically optimizing the microneedle geometrical parameters, array arrangement, and preparation process parameters, we determined the optimal ratio of drug-carrying matrix as 15% PVP (polyvinyl pyrrolidone) K90. The microneedles exhibited significant drug loading gradients, with mean content of (209.99±27.56) μg/patch, (405.31±30.31) μg/patch, and (621.61±34.43) μg/patch. They showed a regular pyramidal structure under SEM and handheld electron microscopy, and their mechanical strength allowed effective penetration into the stratum corneum. The surface contact angles were all < 90°, indicating excellent hydrophilicity. The microneedles dissolved completely within 10 min after skin insertion, achieving a cumulative release rate of 90% (Higuchi model, r=0.996) during 2 hours of in vitro transdermal permeation. The cytotoxicity test and hemolysis test verified good biocompatibility. Pharmacodynamic evaluation showed that the microneedle group demonstrated pain-relieving effect within 15 min, with the pain threshold at the time point of 60 min being 3 times that in the transdermal cream group. The microneedle system developed in this study not only offers an efficient drug delivery option for patients but also establishes an innovative platform for rapid percutaneous delivery of hydrophilic drugs, demonstrating significant potential in perioperative pain management.
Dexmedetomidine/pharmacokinetics* ; Printing, Three-Dimensional ; Needles ; Drug Delivery Systems/methods* ; Administration, Cutaneous ; Animals ; Microinjections/instrumentation* ; Skin Absorption ; Skin/metabolism*

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.
APOBEC-1 Deaminase ; genetics ; metabolism ; Animals ; Bacterial Proteins ; genetics ; metabolism ; Base Sequence ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cytidine ; genetics ; metabolism ; Embryo Transfer ; Embryo, Mammalian ; Endonucleases ; genetics ; metabolism ; Gene Editing ; methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Mice ; Mice, Inbred C57BL ; Microinjections ; Plasmids ; chemistry ; metabolism ; Point Mutation ; RNA, Guide ; genetics ; metabolism ; Thymidine ; genetics ; metabolism ; Zygote ; growth & development ; metabolism ; transplantation

Alzheimer's disease (AD) is an age-related, progressive neurodegenerative disorder that occurs gradually and results in memory, behavior, and personality changes. Abnormal sphingolipid metabolism was reported in AD previously. This study aimed to investigate whether sphK1 could exacerbate the accumulation of amyloid protein (Aβ) and sharpen the learning and memory ability of the animal model of AD using siRNA interference. An adenovirus vector expressing small interfering RNA (siRNA) against the sphK1 gene (sphK1-siRNA) was designed, and the effects of sphK1-siRNA on the APP/PS1 mouse four weeks after treatment with sphK1-siRNA hippocampal injection were examined. SphK1 protein expression was confirmed by using Western blotting and ceramide content coupled with S1P secretion was evaluated by enzyme-linked immunosorbent assay (ELISA). Aβ load was detected by immunohistochemical staining and ELISA. Morris water maze was adopted to test the learning and memory ability of the APP/PS1 mice. A significant difference in the expression of sphK1 protein and mRNA was observed between the siRNA group and the control group. Aβ load in transfected mice was accelerated in vivo, with significant aggravation of the learning and memory ability. The sphK1 gene modulation in the Aβ load and the learning and memory ability in the animal model of AD may be important for the treatment of AD.
Alzheimer Disease ; diagnosis ; physiopathology ; therapy ; Animals ; Disease Models, Animal ; Gene Silencing ; Genetic Therapy ; methods ; Learning Disorders ; diagnosis ; physiopathology ; therapy ; Mice ; Mice, Transgenic ; Microinjections ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; RNA, Small Interfering ; administration & dosage ; genetics ; therapeutic use ; Treatment Outcome

OBJECTIVETo investigate the possibility of repairing spinal cord injury by bone marrow stromal cell (MSC) transplantation and microinjection of chondroitinase ABC (ChABC) in adult rats.

METHODSMSCs isolated from the femoral and tibial bones of new-born Wistar rats were cultured and the cell density was adjusted to 1×10(5)µl before transplantation. SD rats with T8 spinal cord crush injury were divided into 4 groups, namely group A (control) and groups B, C and D with injections of the MSCs, ChABC and MSCs+ChABC at the injury site, respectively. At 0 h, 1 day, 2 days, 3 days, 1 week, and 2 weeks after the injury, the BBB score system was used to evaluate the motion function. At 14 days after the injury, the maximal transverse diameter and actual area of necrosis were evaluated on HE stained sections. GFAP-CS56, GFAP-GAP43 and GFAP-NF160 immunofluorescence double labeling staining were carried out to evaluate the regeneration of the nerve fibers.

RESULTSAt the 14th day after the injury, BBB scores showed significant differences between group A and groups B, C and D (P<0.05), between the groups B and D and between groups C and D, butnot between groups B and C. HE staining showed cavity formation at the injury site in each group, with significant differences between group A and groups B, C and D and also between the latter 3 groups. Immunofluorescence staining revealed more intense expression of GFAP in group A than in the other groups with apparent cavity formation at the lesion site, which was only moderate in groups B and C. The expression of GAP-43 was more intense in group D than in groups B and C. The expression of NF-160 was more intense in group D than in the other 3 groups.

CONCLUSIONThe strategy of MSC transplantation combined with ChABC can be effective for repairing spinal cord injury in adult rats.

Animals ; Bone Marrow Transplantation ; methods ; Chondroitin ABC Lyase ; administration & dosage ; Combined Modality Therapy ; Male ; Microinjections ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; therapy ; Stromal Cells ; transplantation

To optimize program of bovine somatic nuclear transfer, we used two different enucleation procedures (by Spindle-view system & Hoechst 33342 staining), two different procedures to introduce donor nuclei (by ooplasm microinjection & electrofusion), and three different group electrofusion parameters (group 1: 1.9 kV/cm, 10 micros, two; group 2: 1.5 kV/cm, 25 micros, two; group 3: 0.6 kV/cm, 100 micros, one) to reconstruct bovine cloned embryos. The cleavation rates and blastocyst development rates of cloned embryos were used to assess the efficiency of different operational procedure. Finally, the best combination of operational procedure, that the spindle-viewer system was used for oocytes enucleating, and donor cell was electrofused into ooplasm by electrical pulse (1.9 kV/cm, 10 micros, two) to reconstruct bovine cloned embryos. Then the excellent blastocysts were transferred to fosters for producing cloned cattle 80 high-quality cloned blastocysts were transferred into 33 fosters, two cloned calves were produced. According to the results, the optimized program could be used to produce cloned cattle.
Animals ; Cattle ; Cell Nucleus ; physiology ; Cloning, Organism ; veterinary ; Embryo Transfer ; methods ; Embryo, Mammalian ; cytology ; physiology ; Female ; Microinjections ; Nuclear Transfer Techniques ; veterinary ; Oocytes ; cytology ; physiology

This study was performed to produce transgenic Korean native goat (Capra hircus) by laparoscopic embryo transfer (ET) to overcome the limitations of ET performed by laparotomy. Transgenic embryos were produced by DNA pronuclear microinjection of in vivo zygotes. The recipient goats were synchronized for estrus by using an introvaginal progesterone devices as a controlled internal drugreleasing insert (CIDR) for 13 days and injection of 400 IU PMSG 48 h before removal of the insert. Embryos were transferred on day 3 and 4 after removal of the insert. Recipient goats were deprived of feed for 48 h, then suspended in a laparotomy cradle at an angle of 45degrees. After obtaining a sufficient pneumoperitoneum, the laparoscope and forceps were inserted abdominally through 5 mm trocar sleeves. Examination of the ovaries and uterus was performed and then 213 embryos were transferred into the oviducts via the infundibula of 76 recipient goats. To compare pregnancy rates, ET was also performed by laparotomy in 82 recipient goats. The pregnancies in the recipient goats were diagnosed by ultrasound on day 30 after embryo transfer. The pregnancy rate with laparoscopic ET was significantly higher than with ET performed by laparotomy (46.1% vs. 28.6%, p < 0.05). In addition, the pregnancy rates were compared between ovulated and non-ovulated ovaries of the recipient goats in the laparoscopic ET group. No significant difference was observed between the pregnancy rates of ovulated and non-ovulated ovaries (41.3% vs. 33.3%, p < 0.05) suggesting that ET may also be possible in non-ovulated recipients through artificial rupture of Graafian follicles. These results suggest that laparoscopic ET is a highly efficient method for the transfer of goat embryos.
Animals ; Animals, Genetically Modified/*embryology ; Embryo Transfer/methods/*veterinary ; Female ; Goats/*genetics/physiology ; Laparoscopy/*veterinary ; Laparotomy/*veterinary ; Microinjections/veterinary ; Oocytes

OBJECTIVETo evaluate the feasibility of establishing transgenic mice by means of seminiferous tubule microinjection and electroporation (EP) in vivo.

METHODSSpecific pathogen-free (SPF) male Kunming mice divided into 4 groups were subjected to microinjection of two different transfection solutions labeled with enhance green fluorescent protein (EGFP) into the seminiferous tubule of the testis, and in one of the two groups receiving the identical transfection solutions, EP in vivo was performed. After two weeks, the male mice of each group were mated with SPF female Kunming mice with superovulation treatment, and PCR coupled with Southern blotting was performed for the offspring mice.

RESULTSThe results of PCR suggested significant difference in the efficiency of exogenous gene integration between the 4 groups (P<0.01), among which group A achieved the greatest efficiency (45%). Southern blotting did not identify significant difference between the 4 groups (P>0.05), but still suggested the highest efficiency in group A (25%).

CONCLUSIONSeminiferous tubule microinjection in conjunction with subsequent EP in vivo can remarkably enhance the integration efficiency of exogenous genes into the host genome, but this new method needs to be further tested for its potential utility in transgenic animal generation.

Animals ; Blotting, Southern ; Cell Line ; DNA ; administration & dosage ; genetics ; Electroporation ; methods ; Female ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Male ; Mice ; Mice, Transgenic ; Microinjections ; Polymerase Chain Reaction ; Seminiferous Tubules ; Transfection ; methods

Taste responses in the parabrachial nucleus (PBN) are significantly affected by stimulation or lesion of the central nucleus of the amygdala (CeA). To examine if the glutamate receptors in the CeA are involved in this modulation, the effects of microinjection of 6-cyano-7-nitro-quinoxaline-2, 3-dione (CNQX), an AMPA receptor antagonist, into the CeA on the activities of PBN taste neurons were observed by using extracellular recording technique. Responses of PBN taste neurons to taste stimuli were observed before and after CNQX administered to the CeA. In general, drug administration produced a time-dependent suppress of the responses in 30% PBN taste neurons, with the firing rates to HCl and QHCl were significantly lowered (P<0.05). According to the best-stimulus category, 40% NaCl-best (6/15), 30% HCl-best (3/10) and 20% QHCl-best (1/5) neurons decreased their responses to at least one basic taste stimulus after CNQX injection. In HCl- and QHCl-best neurons, the main responses were significantly inhibited after drug injections (P<0.01). The correlation coefficient of responses between the NaCl and the other three tastants decreased after drug administration to the CeA. These results suggest that AMPA receptors within the CeA may be involved in the descending modulation in the PBN taste neurons.
6-Cyano-7-nitroquinoxaline-2,3-dione ; pharmacology ; Amygdala ; drug effects ; physiology ; Animals ; Electric Stimulation ; methods ; Evoked Potentials ; physiology ; Male ; Microinjections ; Pons ; physiology ; Rats ; Rats, Sprague-Dawley ; Receptors, AMPA ; antagonists & inhibitors ; Taste ; physiology ; Taste Threshold

This study was carried out to examine the effect of different donor cell type and micro-manipulation on the development of reconstituted embryos. Cultured mural cumulus cells or fibroblast cells from an adult transgenic goat expressing human erythropoietin(rhEPO) were used as the donor cells in nuclear transfer experiments. The reconstituted eggs were generated by transferring fibroblast cells or cumulus cells into the perivitelline space of enucleated M II oocytes and then followed by electrofusion and activation. After 6 days' incubation in vivo, the reconstructed embryos developed into morulae or blastocysts were transferred into 6 foster recipients. Two of the foster-mothers were pregnant and gave birth to two offspring, which were derived from the fibroblast cell and cumulus cell, respectively. Fingerprint analysis showed that the PCR-RFLP patterns of the two offspring were identical to that of donor goats. PCR results indicated that these cloned goats carried hEPO gene as same as their donor cells.
Animals ; Animals, Genetically Modified ; genetics ; Cell Fusion ; methods ; Cloning, Organism ; Embryo Transfer ; trends ; Erythropoietin ; biosynthesis ; genetics ; Fibroblasts ; cytology ; Goats ; embryology ; genetics ; Humans ; Microinjections ; methods ; Nuclear Transfer Techniques ; Oocytes ; cytology