Positron Emission Tomography Studies of the Biodistribution, Translocation, and Fate of Poly Allyl Amine-Based Carriers for Sirna Delivery by Systemic and Intratumoral Administration.

Polyamine-based vectors offer many advantages for gene therapy, but they are hampered by a limited knowledge on their biological fate and efficacy for nucleic acid delivery. The 18 F radiolabeled siRNA is complexed with poly(allyl amine) hydrochloride (PAH), PEGylated PAH (PAHPEG ), or oleic acid-modified PAH (PAHOleic ) to form polyplexes, and injected them intravenously into healthy rodents. The biodistribution patterns obtained by positron emission tomography (PET) imaging vary according to the polymer used for complexation. Free siRNA is quickly eliminated through the bladder. PAH and oleic acid modify PAH polyplexes accumulate in the lungs and liver. No elimination through the bladder is observed for PAH and PAHOleic within 2 h after administration. PAHPEG polyplexes accumulate in kidneys and are eliminated through the bladder. Polyplexes prepared with 18 F-labeled oleic acid-modified PAH and non-labeled siRNA show similar biodistribution to those prepared with labeled siRNA, but with more accumulation in the lungs due to the presence of non-complexed polymer. Intravenous administration of PAHOleic polyplexes in tumor models results in a limited availability of siRNA. When PAHOleic polyplexes are administered intratumorally in tumor bearing rodents, ≈40% of the radioactivity is retained in the tumor after 180 min while free siRNA is completely eliminated.

Muscle Quality Index is inversely associated with psychosocial variables among Chilean adolescents.

A good muscle quality index (MQI) may have an inverse relationship with psychosocial variables of depression, anxiety, and stress in adolescents. Unfortunately, little scientific evidence has related MQI to psychosocial variables in this population. Therefore, this research aimed to determine the relationship between the MQI and psychosocial variables of depression, anxiety, and stress in Chilean adolescents. In this quantitative correlational design study, sixty adolescents participated voluntarily (mean ± standard deviation [SD]: age 15.11 ± 1.78 years). Anthropometric parameters, prehensile strength, MQI, and psychosocial variables were evaluated. The results showed that adolescents with high levels of MQI presented lower levels of depression (7.50 ± 6.06 vs. 10.97 ± 5.94), anxiety (5.64 ± 4.81 vs. 9.66 ± 5.12), and stress (6.79 ± 5.09 vs. 10 ± 5.58), in addition to reported lower abdominal obesity (WtHR, 0.47 ± 0.07 vs. 0.52 ± 0.07) than those with low levels of MQI. The group with high levels of MQI reported a higher prevalence of nonanxiety (81.3%, p = 0.031) and a lower prevalence of abdominal obesity (55.8%, p = 0.023). Likewise, a significant inverse association was evidenced between MQI and depression (ß; -6.18, 95% CI; -10.11: -2.25, p = 0.003), anxiety (ß; -6.61, 95% CI; -9.83: -3.39, p < 0.001) and stress (ß; -4.90, 95% CI; -8.49: -1.32 p = 0.008). In conclusion, the results suggest that high levels of MQI are associated with a higher prevalence of nonanxiety in adolescents and a significant inverse association between MQI and levels of depression, anxiety, and stress.

On-Demand Chemomagnetic Modulation of Striatal Neurons Facilitated by Hybrid Magnetic Nanoparticles.

Minimally invasive manipulation of cell signaling is critical in basic neuroscience research and in developing therapies for neurological disorders. Here, we describe a wireless chemomagnetic neuromodulation platform for the on-demand control of primary striatal neurons that relies on nanoscale heating events. Iron oxide magnetic nanoparticles (MNPs) are functionally coated with thermoresponsive poly (oligo (ethylene glycol) methyl ether methacrylate) (POEGMA) brushes loaded with dopamine. Dopamine loaded MNPs-POEGMA are co-cultured with primary striatal neurons. When alternating magnetinec fields (AMF) are applied, MNPs undergo hysteresis power loss and dissipate heat. The local heat produced by MNPs initiates a thermodynamic phase transition on POEGMA brushes resulting in polymer collapse and dopamine release. AMF-triggered dopamine release enhances the response of dopamine ion channels expressed on the cell membranes enhancing the activity of ~50% of striatal neurons subjected to the treatment. Chemomagnetic actuation on dopamine receptors is confirmed by blocking D1 and D2 receptors. The reversible thermodynamic phase transition of POEGMA brushes allow the on-demand release of dopamine in multiple microdoses. AMF-triggered dopamine release from MNPs-POEGMA causes no cell cytotoxicity nor promotes cell ROS production. This research represents a fundamental step forward for the chemomagnetic control of neural activity using hybrid magnetic nanomaterials with tailored physical properties.

Phase transition characterization of poly(oligo(ethylene glycol)methyl ether methacrylate) brushes using the quartz crystal microbalance with dissipation.

Heterogeneous non-linear poly(ethylene glycol) analogs, like poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), are of particular interest in the fabrication of smart biocompatible coatings as they undergo a reversible macromolecular rearrangement in response to external heat stimuli. The phase transition dynamics of POEGMA coatings in response to external temperature stimuli have been poorly investigated. The quartz crystal microbalance with dissipation (QCM-D) can be used to investigate the phase transition of these functional coatings as polymer brushes in a dynamic and noninvasive in situ measurement. POEGMA brushes with different thickness are synthesized from the surface of a QCM-D sensor following a living radical polymerization technique by varying the monomer molecular weight. Investigations on the thermoresponsive collapse and swelling of POEGMA brushes grafted from the surface of a QCM-D sensor reveal the reversible phase transition nature of these coatings. Furthermore, the potential of these smart coatings in the field of biotechnology was explored by investigating the absorption and desorption of a model drug. A pulsatile drug release profile triggered by an increase in temperature is observed from POEGMA brushes. POEGMA brushes have the potential to be utilized as polymer coatings for controlled and programable drug release.

Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 ß-xylosidase from G. stearothermophilus.

Cross-linked enzyme aggregates (CLEAs) of the Y509E mutant of glycoside hydrolase family 52 ß-xylosidase from Geobacillus stearothermophilus with dual activity of ß-xylosidase and xylanase (XynB2Y509E) were prepared. Ammonium sulfate was used as the precipitant agent, and glutaraldehyde as cross-linking agent. The optimum conditions were found to be 90% ammonium sulfate, 12.5 mM glutaraldehyde, 3 h of cross-linking reaction at 25 °C, and pH 8.5. Under these (most effective) conditions, XynB2Y509E-CLEAs retained 92.3% of their original ß-xylosidase activity. Biochemical characterization of both crude and immobilized enzymes demonstrated that the maximum pH and temperature after immobilization remained unchanged (pH 6.5 and 65 °C). Moreover, an improvement in pH stability and thermostability was also found after immobilization. Analysis of kinetic parameters shows that the K m value of XynB2Y509E-CLEAs obtained was slightly higher than that of free XynB2Y509E (1.2 versus 0.9 mM). Interestingly, the xylanase activity developed by the mutation was also conserved after the immobilization process.

Adolescent Depression: Differential Symptom Presentations in Deaf and Hard-of-Hearing Youth Using the Patient Health Questionnaire-9.

The present study examined differences in symptom presentation in screening for pediatric depression via evaluation of the Patient Health Questionnaire-9 (PHQ-9). In particular, we examined whether PHQ-9 items function differentially among deaf and hard-of-hearing (DHH; n = 75) and hearing (n = 75) youth based on participants recruited from crisis assessment services. Multiple indicators multiple causes models were used to examine whether items of the PHQ-9 functioned differently between groups as well as whether there were group differences in the mean severity of depressive symptoms. Results indicate that DHH youth were more likely to endorse psychosomatic items, and less likely to endorse an affective item. These findings indicate that the PHQ-9 functions differently when used with DHH youth. Implications of these findings are discussed, including both for future work with the PHQ-9 and with regard to the conceptualization of depression across hearing groups.

The relationship of perceived campus culture to mental health help-seeking intentions.

Despite mental health issues being widespread on college campuses, the majority of college students do not seek help. Prior research suggests several individual factors that may be related to mental health help-seeking including age, gender, and prior treatment experience. However, there has been little work considering the broader role of the college environment on person-level predictors of mental health help-seeking, specifically the relationship with perceived campus culture. Thus, informed by the theory of planned behavior (Ajzen, 1991), the purpose of this study was to examine the relationship between perceived campus cultural perspectives on different personal processes, such as attitudes toward treatment, stigma, and treatment barriers that are believed to relate to mental health help-seeking intentions. Participants were 212 undergraduate students from a large university in the southeastern United States. As hypothesized, we found a significant mediation relationship for personal attitudes in the relationship between perceived campus attitudes and help-seeking intentions. In contrast, analyses did not support mediation relationships for personal barriers or personal stigma. These findings suggest that perceived campus culture may serve an important role in personal mental health treatment beliefs. Campus mental health policies and prevention programming may consider targeting perceived campus culture as an important means for increasing personal positive beliefs toward mental health treatment. (PsycINFO Database Record

Characterization of molecular transport in ultrathin hydrogel coatings for cellular immunoprotection.

PEG hydrogels are routinely used in immunoprotection applications to hide foreign cells from a host immune system. Size-dependent transport is typically exploited in these systems to prevent access by macromolecular elements of the immune system while allowing the transport of low molecular weight nutrients. This work studies a nanoscale hydrogel coating for improved transport of beneficial low molecular weight materials across thicker hydrogel coatings while completely blocking transport of undesired larger molecular weight materials. Coatings composed of PEG diacrylate of molecular weight 575 and 3500 Da were studied by tracking the transport of fluorescently labeled dextrans across the coatings. The molecular weight of dextran at which the transport is blocked by these coatings are consistent with cutoff values in analogous bulk PEG materials. Additionally, the diffusion constants of 4 kDa dextrans across PEG 575 coatings (9.5 × 10(-10)-2.0 × 10(-9) cm(2)/s) was lower than across PEG 3500 coatings (5.9-9.8 × 10(-9) cm(2)/s), and these trends and magnitudes agree with bulk scale models. Overall, these nanoscale thin PEG diacrylate films offer the same size selective transport behavior of bulk PEG diacrylate materials, while the lower thickness translates directly to increased flux of beneficial low molecular weight materials.

Effects of a Low Dose of Orally Administered Creatine Monohydrate on Post-Fatigue Muscle Power in Young Soccer Players.

The use of creatine monohydrate (Cr) in professional soccer is widely documented. However, the effect of low doses of Cr on the physical performance of young soccer players is unknown. This study determined the effect of a low dose of orally administered Cr on muscle power after acute intra-session fatigue in young soccer players. Twenty-eight young soccer players (mean age = 17.1 ± 0.9 years) were randomly assigned to either a Cr (n = 14, 0.3 g·kg-1·day-1 for 14 days) or placebo group (n = 14), using a two-group matched, double-blind, placebo-controlled design. Before and after supplementation, participants performed 21 repetitions of 30 m (fatigue induction), and then, to measure muscle power, they performed four repetitions in half back squat (HBS) at 65% of 1RM. Statistical analysis included a two-factor ANOVA (p ˂ 0.05). Bar velocity at HBS, time: p = 0.0006, ŋp2 = 0.22; group: p = 0.0431, ŋp2 = 0.12, time × group p = 0.0744, ŋp2 = 0.02. Power at HBS, time: p = 0.0006, ŋp2 = 0.12; group: p = 0.16, ŋp2 = 0.06, time × group: p = 0.17, ŋp2 = 0.009. At the end of the study, it was found that, after the induction of acute intra-session fatigue, a low dose of Cr administered orally increases muscle power in young soccer players.

Self-Assembled Oleic Acid-Modified Polyallylamines for Improved siRNA Transfection Efficiency and Lower Cytotoxicity.

Small interference RNA (siRNA) is a tool for gene modulation, which can silence any gene involved in genetic disorders. The potential of this therapeutic tool is hampered by RNA instability in the blood stream and difficulties to reach the cytosol. Polyamine-based nanoparticles play an important role in gene delivery. Polyallylamine hydrochloride (PAH) is a polycation displaying primary amines that can be easily chemically modified to match the balance between cell viability and siRNA transfection. In this work, PAH has been covalently functionalized with oleic acid at different molar ratios by carbodiimide chemistry. The substituted polymers form polyplexes that keep positive surface charge and fully encapsulate siRNA. Oleic acid substitution improves cell viability in the pulmonary cell line A549. Moreover, 6 and 14% of oleic acid substitution show an improvement in siRNA transfection efficiency. CD47 is a ubiquitous protein which acts as "don't eat me signal." SIRPα protein of macrophages recognizes CD47, leading to tumor cell phagocytosis by macrophages. By knocking down CD47 with siRNA, cancer cells become vulnerable to be eliminated by the immune system. PAH-oleic acid substitutes show high efficacy in silencing the CD47 protein, making them a potential candidate for immunotherapy.

Elucidating Mechanotransduction Processes During Magnetomechanical Neuromodulation Mediated by Magnetic Nanodiscs.

Purpose: Noninvasive cell-type-specific manipulation of neural signaling is critical in basic neuroscience research and in developing therapies for neurological disorders. Magnetic nanotechnologies have emerged as non-invasive neuromodulation approaches with high spatiotemporal control. We recently developed a wireless force-induced neurostimulation platform utilizing micro-sized magnetic discs (MDs) and low-intensity alternating magnetic fields (AMFs). When targeted to the cell membrane, MDs AMFs-triggered mechanoactuation enhances specific cell membrane receptors resulting in cell depolarization. Although promising, it is critical to understand the role of mechanical forces in magnetomechanical neuromodulation and their transduction to molecular signals for its optimization and future translation. Methods: MDs are fabricated using top-down lithography techniques, functionalized with polymers and antibodies, and characterized for their physical properties. Primary cortical neurons co-cultured with MDs and transmembrane protein chemical inhibitors are subjected to 20 s pulses of weak AMFs (18 mT, 6 Hz). Calcium cell activity is recorded during AMFs stimulation. Results: Neuronal activity in primary rat cortical neurons is evoked by the AMFs-triggered actuation of targeted MDs. Ion channel chemical inhibition suggests that magnetomechanical neuromodulation results from MDs actuation on Piezo1 and TRPC1 mechanosensitive ion channels. The actuation mechanisms depend on MDs size, with cell membrane stretch and stress caused by the MDs torque being the most dominant. Conclusions: Magnetomechanical neuromodulation represents a tremendous potential since it fulfills the requirements of negligible heating (ΔT < 0.1 °C) and weak AMFs (< 100 Hz), which are limiting factors in the development of therapies and the design of clinical equipment. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00786-8.

Efficiency Assessment between Entrapment and Covalent Bond Immobilization of Mutant ß-Xylosidase onto Chitosan Support.

The Y509E mutant of ß-xylosidase from Geobacillus stearothermophilus (XynB2Y509E) (which also bears xylanase activity) has been immobilized in chitosan spheres through either entrapment or covalent bond formation methods. The maximum immobilization yield by entrapment was achieved by chitosan beads developed using a 2% chitosan solution after 1 h of maturation time in CFG buffer with ethanol. On the other hand, the highest value in covalent bond immobilization was observed when employing chitosan beads that were prepared from a 2% chitosan solution after 4 h of activation in 1% glutaraldehyde solution at pH 8. The activity expressed after immobilization by covalent bonding was 23% higher compared to the activity expressed following entrapment immobilization, with values of 122.3 and 99.4 IU.g-1, respectively. Kinetic data revealed that catalytic turnover values were decreased as compared to a free counterpart. Both biocatalysts showed increased thermal and pH stability, along with an improved storage capacity, as they retained 88% and 40% of their activity after being stored at 4 °C for two months. Moreover, XynB2Y509E immobilized by covalent binding also exhibited outstanding reusability, retaining 92% of activity after 10 cycles of reuse. In conclusion, our results suggest that the covalent bond method appears to be the best choice for XynB2Y509E immobilization.

Poly (ß-amino Ester) Nanoparticles Modified with a Rabies Virus-derived peptide for the Delivery of ASCL1 Across a 3D In Vitro Model of the Blood Brain Barrier.

Gene editing has emerged as a therapeutic approach to manipulate the genome for killing cancer cells, protecting healthy tissues, and improving immune response to a tumor. The gene editing tool achaete-scute family bHLH transcription factor 1 CRISPR guide RNA (ASCL1-gRNA) is known to restore neuronal lineage potential, promote terminal differentiation, and attenuate tumorigenicity in glioblastoma tumors. Here, we fabricated a polymeric nonviral carrier to encapsulate ASCL1-gRNA by electrostatic interactions and deliver it into glioblastoma cells across a 3D in vitro model of the blood-brain barrier (BBB). To mimic rabies virus (RV) neurotropism, gene-loaded poly (ß-amino ester) nanoparticles are surface functionalized with a peptide derivative of rabies virus glycoprotein (RVG29). The capability of the obtained NPs, hereinafter referred to as RV-like NPs, to travel across the BBB, internalize into glioblastoma cells and deliver ASCL1-gRNA are investigated in a 3D BBB in vitro model through flow cytometry and CLSM microscopy. The formation of nicotinic acetylcholine receptors in the 3D BBB in vitro model is confirmed by immunochemistry. These receptors are known to bind to RVG29. Unlike Lipofectamine that primarily internalizes and transfects endothelial cells, RV-like NPs are capable to travel across the BBB, preferentially internalize glioblastoma cells and deliver ASCL1-gRNA at an efficiency of 10 % causing non-cytotoxic effects.

Soft nano and microstructures for the photomodulation of cellular signaling and behavior.

Photoresponsive soft materials are everywhere in the nature, from human's retina tissues to plants, and have been the inspiration for engineers in the development of modern biomedical materials. Light as an external stimulus is particularly attractive because it is relatively cheap, noninvasive to superficial biological tissues, can be delivered contactless and offers high spatiotemporal control. In the biomedical field, soft materials that respond to long wavelength or that incorporate a photon upconversion mechanism are desired to overcome the limited UV-visible light penetration into biological tissues. Upon light exposure, photosensitive soft materials respond through mechanisms of isomerization, crosslinking or cleavage, hyperthermia, photoreactions, electrical current generation, among others. In this review, we discuss the most recent applications of photosensitive soft materials in the modulation of cellular behavior, for tissue engineering and regenerative medicine, in drug delivery and for phototherapies.

Modulating cell signalling in vivo with magnetic nanotransducers.

Weak magnetic fields offer nearly lossless transmission of signals within biological tissue. Magnetic nanomaterials are capable of transducing magnetic fields into a range of biologically relevant signals in vitro and in vivo. These nanotransducers have recently enabled magnetic control of cellular processes, from neuronal firing and gene expression to programmed apoptosis. Effective implementation of magnetically controlled cellular signalling relies on careful tailoring of magnetic nanotransducers and magnetic fields to the responses of the intended molecular targets. This primer discusses the versatility of magnetic modulation modalities and offers practical guidelines for selection of appropriate materials and field parameters, with a particular focus on applications in neuroscience. With recent developments in magnetic instrumentation and nanoparticle chemistries, including those that are commercially available, magnetic approaches promise to empower research aimed at connecting molecular and cellular signalling to physiology and behaviour in untethered moving subjects.

Managing beyond ecosystem limits at the land-sea interface: The case of sandy beaches.

Sandy beaches are part of an integral social-ecological system whose management has to encompass the natural and societal features of the catchment and the adjacent marine area, as well as the beach itself. Using a multi-use and complex beach system in Uruguay, the La Coronilla and Barra del Chuy resort, we interrogate those natural and societal features by employing the DAPSI(W)R(M) cause-consequence-response cycle and pathways. This identifies the Drivers, Activities, Pressures, State change on the natural system, Impacts (on the Welfare of the human system), and the Responses (requiring management Measures). We contend that this approach is needed for the sustainable development and use of this ecosystem and its biodiversity protection. This also indicates the importance of a holistic and systems approach, which is necessary, valid and valuable for sandy beaches worldwide.

DSCAM is differentially patterned along the optic axon pathway in the developing Xenopus visual system and guides axon termination at the target.

BACKGROUND: The Xenopus retinotectal circuit is organized topographically, where the dorsal-ventral axis of the retina maps respectively on to the ventral-dorsal axis of the tectum; axons from the nasal-temporal axis of the retina project respectively to the caudal-rostral axis of the tectum. Studies throughout the last two decades have shown that mechanisms involving molecular recognition of proper termination domains are at work guiding topographic organization. Such studies have shown that graded distribution of molecular cues is important for topographic mapping. However, the complement of molecular cues organizing topography along the developing optic nerve, and as retinal axons cross the chiasm and navigate towards and innervate their target in the tectum, remains unknown. Down syndrome cell adhesion molecule (DSCAM) has been characterized as a key molecule in axon guidance, making it a strong candidate involved in the topographic organization of retinal fibers along the optic path and at their target. METHODS: Using a combination of whole-brain clearing and immunohistochemistry staining techniques we characterized DSCAM expression and the projection of ventral and dorsal retinal fibers starting from the eye, following to the optic nerve and chiasm, and into the terminal target in the optic tectum in Xenopus laevis tadpoles. We then assessed the effects of DSCAM on the establishment of retinotopic maps through spatially and temporally targeted DSCAM knockdown on retinal ganglion cells (RGCs) with axons innervating the optic tectum. RESULTS: Highest expression of DSCAM was localized to the ventral posterior region of the optic nerve and chiasm; this expression pattern coincides with ventral fibers derived from ventral RGCs. Targeted downregulation of DSCAM expression on ventral RGCs affected the segregation of medial axon fibers from their dorsal counterparts within the tectal neuropil, indicating that DSCAM plays a role in retinotopic organization. CONCLUSION: These findings together with previous studies demonstrating cell-autonomous roles for DSCAM during the development of pre- and postsynaptic arbors in the Xenopus retinotectal circuit indicates that DSCAM exerts multiple roles in coordinating axon targeting and structural connectivity in the developing vertebrate visual system.

Wireless Force-Inducing Neuronal Stimulation Mediated by High Magnetic Moment Microdiscs.

Noninvasive manipulation of cell signaling is critical in basic neuroscience research and in developing therapies for neurological disorders and psychiatric conditions. Here, the wireless force-induced stimulation of primary neuronal circuits through mechanotransduction mediated by magnetic microdiscs (MMDs) under applied low-intensity and low-frequency alternating magnetic fields (AMFs), is described. MMDs are fabricated by top-down lithography techniques that allow for cost-effective mass production of biocompatible MMDs with high saturation and zero magnetic magnetic moment at remanence. MMDs are utilized as transducers of AMFs into mechanical forces. When MMDs are exposed to primary rat neuronal circuits, their magneto-mechanical actuation triggers the response of specific mechanosensitive ion channels expressed on the cell membranes activating ≈50% of hippocampal and ≈90% of cortical neurons subjected to the treatment. Mechanotransduction is confirmed by the inhibition of mechanosensitive transmembrane channels with Gd3+ . Mechanotransduction mediated by MMDs cause no cytotoxic effect to neuronal cultures. This technology fulfills the requirements of cell-type specificity and weak magnetic fields, two limiting factors in the development of noninvasive neuromodulation therapies and clinical equipment design. Moreover, high efficiency and long-lasting stimulations are successfully achieved. This research represents a fundamental step forward for magneto-mechanical control of neural activity using disc-shaped micromaterials with tailored magnetic properties.

Under the Surface: The Role of Covert Cues in Peer Suicide Risk Referrals.

Suicidal thoughts and behaviors are highly prevalent among adolescents, and peers are often the first, and sometimes only, people to know about youth suicidality. Since many adolescents do not directly disclose suicidal thoughts, school-based suicide prevention programs aim to train youth to recognize warning signs of suicide in their peers that serve as "cues" to refer at-risk peers to an appropriate adult. However, peer-presented cues vary widely in presentation, and adolescents are more likely to recognize overt (i.e., obvious or explicit) as opposed to covert (i.e., hidden or implied) cues. The type of cue exhibited may, in turn, affect whether adolescents make a referral to an adult. The current study examined whether training suicide prevention influences referral intentions for overt and covert suicide cues. Participants included 244 high school students (54% female; M age = 16.21) in the Southeastern United States who received suicide prevention training (SOS; Signs of Suicide) as part of their health curriculum. Prior to training, students endorsed higher referral intentions for peers exhibiting overt compared to covert cues. Training was associated with increased intentions to refer peers across cue type, but referral intentions for covert cues improved significantly from pre to post-training while those for overt cues remained high and stable. Findings suggest that suicide prevention training might differentially improve students' ability to detect and respond appropriately to less obvious indicators of suicide risk. These findings may inform the adaptation and development of future, more nuanced school-based suicide prevention programming.

Stabilization of Poly (ß-Amino Ester) Nanoparticles for the Efficient Intracellular Delivery of PiggyBac Transposon.

The administration of gene-editing tools has been proposed as a promising therapeutic approach for correcting mutations that cause diseases. Gene-editing tools, composed of relatively large plasmid DNA constructs that often need to be co-delivered with a guiding protein, are unable to spontaneously penetrate mammalian cells. Although viral vectors facilitate DNA delivery, they are restricted by the size of the plasmid to carry. In this work, we describe a strategy for the stable encapsulation of the gene-editing tool piggyBac transposon into Poly (ß-amino ester) nanoparticles (NPs). We propose a non-covalent and a covalent strategy for stabilization of the nanoformulation to slow down release kinetics and enhance intracellular delivery. We found that the formulation prepared by covalently crosslinking Poly (ß-amino ester) NPs are capable to translocate into the cytoplasm and nuclei of human glioblastoma (U87MG) cells within 1 h of co-culturing, without the need of a targeting moiety. Once internalized, the nanoformulation dissociates, delivering the plasmid presumably as a response to the intracellular acidic pH. Transfection efficiency is confirmed by green fluorescence protein (GFP) expression in U87MG cells. Covalently stabilized Poly (ß-amino ester) NPs are able to transfect ~55% of cells causing non-cytotoxic effects. The strategy described in this work may serve for the efficient non-viral delivery of other gene-editing tools.