The Potential of PIP3 in Enhancing Wound Healing.

Given the role of phosphatidylinositol 3,4,5-trisphosphate (PIP3) in modulating cellular processes such as proliferation, survival, and migration, we hypothesized its potential as a novel therapeutic agent for wound closure enhancement. In this study, PIP3 was examined in its free form or as a complex with cationic starch (Q-starch) as a carrier. The intracellular bioactivity and localization of free PIP3 and the Q-starch/PIP3 complexes were examined. Our results present the capability of Q-starch to form complexes with PIP3, facilitate its cellular membrane internalization, and activate intracellular paths leading to enhanced wound healing. Both free PIP3 and Q-starch/PIP3 complexes enhanced monolayer gap closure in scratch assays and induced amplified collagen production within HaCAT and BJ fibroblast cells. Western blot presented enhanced AKT activation by free or complexed PIP3 in BJ fibroblasts in which endogenous PIP3 production was pharmacologically inhibited. Furthermore, both free PIP3 and Q-starch/PIP3 complexes expedited wound closure in mice, after single or daily dermal injections into the wound margins. Free PIP3 and the Q-starch/PIP3 complexes inherently activated the AKT signaling pathway, which is responsible for crucial wound healing processes such as migration; this was also observed in wound assays in mice. PIP3 was identified as a promising molecule for enhancing wound healing, and its ability to circumvent PI3K inhibition suggests possible implications for chronic wound healing.

Cargo-Dependent Targeted Cellular Uptake Using Quaternized Starch as a Carrier.

The tailored design of drug delivery systems for specific therapeutic agents is a prevailing approach in the field. In this paper, we present a study that highlights the potential of our modified starch, Q-starch, as a universal and adaptable drug delivery carrier for diverse therapeutic agents. We investigate the ability of Q-starch/cargo complexes to target different organelles within the cellular landscape, based on the specific activation sites of therapeutic agents. Plasmid DNA (pDNA), small interfering RNA (siRNA), and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) were chosen as representative therapeutic molecules, acting in the nucleus, cytoplasm, and membrane, respectively. By carrying out comprehensive characterizations, employing dynamic light scattering (DLS), determining the zeta potential, and using cryo-transmitting electron microscopy (cryo-TEM), we reveal the formation of nano-sized, positively charged, and spherical Q-starch complexes. Our results demonstrate that these complexes exhibit efficient cellular uptake, targeting their intended organelles while preserving their physical integrity and functionality. Notably, the intracellular path of the Q-starch/cargo complex is guided by the cargo itself, aligning with its unique biological activity site. This study elucidates the versatility and potency of Q-starch as a versatile drug delivery carrier, paving the way for novel applications offering targeted delivery strategies for potential therapeutic molecules.

Cell stiffness predicts cancer cell sensitivity to ultrasound as a selective superficial cancer therapy.

We hypothesize that the biomechanical properties of cells can predict their viability, with Young's modulus representing the former and cell sensitivity to ultrasound representing the latter. Using atomic force microscopy, we show that the Young's modulus stiffness measure is significantly lower for superficial cancer cells (squamous cell carcinomas and melanoma) compared with noncancerous keratinocyte cells. In vitro findings reveal a significant difference between cancerous and noncancerous cell viability at the four ultrasound energy levels evaluated, with different cell lines exhibiting different sensitivities to the same ultrasound intensity. Young's modulus correlates with cell viability (R 2 = 0.93), indicating that this single biomechanical property can predict cell sensitivity to ultrasound treatment. In mice, repeated ultrasound treatment inhibits tumor growth without damaging healthy skin tissue. Histopathological tumor analysis indicates ultrasound-induced focal necrosis at the treatment site. Our findings provide a strong rationale for developing ultrasound as a noninvasive selective treatment for superficial cancers.

Tailor-Made Single-Core PLGA Microbubbles as Acoustic Cavitation Enhancers for Therapeutic Applications.

Microbubbles (MBs), being gas bubbles encapsulated inside a solid shell, have been investigated extensively in the field of therapeutic ultrasound as acoustic cavitation enhancers. Hard-shell MBs have an advantage over soft-shell MBs due to their improved stability. Poly(lactic-co-glycolic acid) (PLGA) is one of the most attractive polymers for hard-shell MB synthesis; however, very little is known regarding the effect of synthesis parameters on the acoustic cavitation activity of PLGA MBs and the tunability of this activity. In this study, by manipulating the synthesis parameters, we were able to control the characteristics of the MBs, such as their internal structure, gas core, size distribution, and shell thickness, which significantly affect the total acoustic cavitation activity that they exhibit (i.e., their cavitation dose). We showed that single-core MBs filled with C3F8 gas can produce cavitation effects for extended periods under continuous circulation. These MBs exhibited high stability, and their cavitation activity was not affected by prior circulation in the system. Preliminary in vivo results demonstrated that intravenously injected MBs did not cause adverse effects and produced cavitation activity that increased the permeability of the pig blood-brain barrier. Although more tests should be performed to evaluate the MB long-term safety and activity in vivo, these encouraging results suggest that our PLGA MBs have potential for future therapeutic applications as cavitation enhancers.

SP-D loaded PLGA nanoparticles as drug delivery system for prevention and treatment of premature infant's lung diseases.

Preterm infants, particularly those who born between 23 and 28 weeks' gestation, suffer from a very high incidence of respiratory distress syndrome (RDS) related to pulmonary immaturity and inability to make Pulmonary Surfactant (PS). These infants are supported by the use of oxygen, ventilators, and routine administration of surfactant replacement. The currently commercial surfactant replacement therapies do not contain hydrophilic surfactant proteins such as Surfactant Protein D (SP-D). These proteins have a key role in the innate lung host defense, thus the development of a sustained release vehicle that provides SP-D for long periods in preterm infants' lungs would exploit the therapeutic potential of SP-D and other pulmonary medications. The proposed SP-D delivery system is based on nanoparticles (NPs) composed of poly (lactic acid-co-glycolic acid) (PLGA), a biodegradable, FDA approved biopolymer. The resulted NPs were spherical with high Zeta potential value, were not toxic to A-549 lungs cells, and did not induce any inflammatory response in mouse's lungs for short and long-term periods. Moreover, SP-D released from NPs showed biological activity for several days and in vivo release experiment of SP-D loaded NPs revealed that SP-D was released from NPs in mouse lungs with different NPs delivery doses.

Ultrasound targeting of Q-starch/miR-197 complexes for topical treatment of psoriasis.

Psoriasis is a common, worldwide autoinflammatory, incurable skin disease. miR-197 has therapeutic potential for psoriasis since it can down-regulate the expression of both IL-22RA1 and IL-17RA, subunits of the receptors of IL-22 and IL-17, respectively, which are key cytokines in the disease. Although miR-197 has the potential to treat the disease, several inherent physical barrier properties of the skin challenge miRNA's delivery to the target skin cells. In the present study, we evaluated a therapeutic approach that combines the use of ultrasound (US) as a means to enhance skin permeability with quaternized starch (Q-starch) as an miRNA delivery carrier. This resulted in decreased expression of the miR-197 target proteins and in a significant reduction in the psoriatic activity markers. Our results demonstrate the potential of combinations of US and Q-starch/miR-197 complexes for the topical skin treatment of psoriasis.

Synthesis, characterization, and self-assembly with plasmid DNA of a quaternary ammonium derivative of pectic galactan and its fluorescent labeling for bioimaging applications.

Quaternized derivatives of pectic galactan (QPG) were synthesized by a reaction of pectic galactan (PG) with 3-chloro-2-hydroxypropyl trimethyl ammonium chloride (CHPTAC) in the presence of aqueous sodium hydroxide solution under mild reaction conditions. The results showed that the concentration of CHPTAC and NaOH has great impact on the quaternization reaction. QPG was found to interact electrostatically with plasmid DNA in aqueous solution to form complexes in globular condensed morphology in a nanometer scale size ranging from 60 to 160nm. Complexes formed with QPG fluorescently labeled with 5-DTAF (QPG-5-DTAF) were introduced to the C6 rat glioma cell line, and were found to be able to enter the cell and approach the nucleus within 24h. The results suggest that this type of modified natural polysaccharide may have an advantage as a biocompatible and biodegradable gene delivery carrier and furthermore may serve as a cell specific carrier.

Polymeric carrier-mediated intracellular delivery of phosphatidylinositol-3,4,5-trisphosphate to overcome insulin resistance.

BACKGROUND: Phosphatidylinositol-3,4,5-trisphosphate (PIP3) is a major lipid second messenger in insulin-mediated signalling towards the metabolic actions of this hormone in muscle and fat. PURPOSE: Assessing the intracellular transport of exogenous PIP3 attached to a polymeric carrier in an attempt to overcome cellular insulin resistance. METHODS: Artificial chromatic bio-mimetic membrane vesicles composed of dimyristoylphosphatidylcholine and polydiacetylene were applied to screen the polymeric carriers. PIP3 cellular localization and bio-activity was assessed by fluorescent and live-cell microscopy in L6 muscle cells and in 3T3-L1 adipocytes. RESULTS AND DISCUSSION: We demonstrate that a specific-branched polyethylenimine (PEI-25, 25 kDa) carrier forms complexes with PIP3 that interact with the bio-mimetic membrane vesicles in a manner predictive of their interaction with cells: In L6 muscle cells, PEI-25/fluorescent-PIP3 complexes are retarded at the cell perimeter. PEI-25/PIP3 complexes retain their bio-activity, engaging signalling steps downstream of PIP3, even in muscle cells rendered insulin resistant by exposure to high glucose/high insulin. CONCLUSIONS: Inducing insulin actions by intracellular PIP3 delivery (PEI-25/PIP3 complexes) in some forms of insulin-resistant cells provides the first proof-of-principle for the potential therapeutic use of PIP3 in a "second-messenger agonist" approach. In addition, utilization of an artificial bio-mimetic membrane platform to screen for highly efficient PIP3 delivery predicts biological function in cells.

Harvesting Low Molecular Weight Biomarkers Using Gold Nanoparticles.

We developed and characterized a platform based on gold (Au) nanoparticles (NPs) coated with poly(acrylic acid) (PAA) for harvesting positively charged, low molecular weight (LMW) proteins. The particles are synthesized using a layer by layer (LbL) procedure: first the gold NPs are coated with positively charged polyethylenimine (PEI) and subsequently with PAA. This simple procedure produces stable PAA-PEI-Au (PPAu) NPs with high selectivity and specificity. PPAu NPs successfully harvested, separated, and detected various LMW proteins and peptides from serum containing a complex mixture of abundant high molecular weight (HMW) proteins, including bovine serum albumin (BSA) and Immunoglobulin G (IgG). In addition, PPAu NPs selectively harvested and separated LMW proteins from serum in the presence of another positively charged competing protein. Furthermore, PPAu NPs successfully harvested a LMW biomarker in a mock diseased state. This system can be applied in various biomedical applications where selective harvesting and identifying of LMW proteins is required. A particularly useful application for this system can be found in early cancer diagnosis as described hereinafter.

Fetal membrane transport enhancement using ultrasound for drug delivery and noninvasive detection.

PURPOSE: The purpose of this research was to evaluate the effect of ultrasound on mass transport across fetal membrane for direct fetal drug delivery and sensing of the amniotic fluid in a noninvasive manner. METHODS: Post-delivery human fetal membranes (chorioamnion) were used for in vitro experiments, in which the effect of ultrasound on transport across fetal membrane of fluorescent model molecule (250 kDa) was evaluated. Ex vivo experiments were carried out on a whole rat amniotic sac. The model molecule or alpha-fetoprotein was injected into the amniotic sac through the placenta. Transport of these molecules across pre- and post-insonation of the amniotic sac was evaluated. The ultrasound enhancement's mechanism was also assessed. RESULTS: The greatest enhancement in mass transport (43-fold) in vitro was achieved for 5 min of insonation (20 kHz, 4.6 W/cm(2), 5 mm distance). Ex vivo results showed a rapid increase (23-fold) in mass transport of the model molecule and also for alphafetoprotein following 30 s of insonation (20 kHz, 4.6 W/cm(2), 5 mm distance). CONCLUSIONS: Mass transport across fetal membranes was enhanced post-insonation both in vitro and ex vivo in a reversible and transient manner. We suggest that exterior (to the amniotic sac) ultrasound-induced cavitation is the main mechanism of action.

Quaternized starch-based carrier for siRNA delivery: from cellular uptake to gene silencing.

RNAi therapeutics is a powerful tool for treating diseases by sequence-specific targeting of genes using siRNA. Since its discovery, the need for a safe and efficient delivery system for siRNA has increased. Here, we have developed and characterized a delivery platform for siRNA based on the natural polysaccharide starch in an attempt to address unresolved delivery challenges of RNAi. Modified potato starch (Q-starch) was successfully obtained by substitution with quaternary reagent, providing Q-starch with cationic properties. The results indicate that Q-starch was able to bind siRNA by self-assembly formation of complexes. For efficient and potent gene silencing we monitored the physical characteristics of the formed nanoparticles at increasing N/P molar ratios. The minimum ratio for complete entrapment of siRNA was 2. The resulting complexes, which were characterized by a small diameter (~30 nm) and positive surface charge, were able to protect siRNA from enzymatic degradation. Q-starch/siRNA complexes efficiently induced P-glycoprotein (P-gp) gene silencing in the human ovarian adenocarcinoma cell line, NCI-ADR/Res (NAR), over expressing the targeted gene and presenting low toxicity. Additionally, Q-starch-based complexes showed high cellular uptake during a 24-hour study, which also suggested that intracellular siRNA delivery barriers governed the kinetics of siRNA transfection. In this study, we have devised a promising siRNA delivery vector based on a starch derivative for efficient and safe RNAi application.

A novel approach for noninvasive drug delivery and sensing through the amniotic sac.

Current invasive prenatal tests (amniocentesis and chorionic villus sampling) are known for their risk to the fetus. In the last decade, the use and awareness of these prenatal tests have increased, resulting in growing demand for a safe, non-invasive, and accurate prenatal test. Chemical penetration enhancers (CPEs) have long been used to increase transport phenomena across skin and other membranes (e.g., tympanic membrane). The amniotic sac membrane is called the chorioamnion (CA) membrane and serves as the physical barrier between the fetus and the mother. In this research, the effect of CPEs on human CA mass transport was evaluated both in vitro and ex vivo. The results show that the tested CPEs exhibit an enhancing effect on CA mass transport. Based on the permeability results, two mechanisms of action were suggested: "extractors" and "fluidizers". Fourier transform infrared (FTIR) and rapid colorimetric screening measurements supported the mechanisms, based on which, more potent compounds were designed and tested for their enhancing effect. The enhancing mass transport effect of CPEs on CA membrane may be used both for sampling of cell-free DNA and for noninvasively administering drugs and other biological agents to the amniotic sac.

Modified pectin-based carrier for gene delivery: cellular barriers in gene delivery course.

The use of polysaccharides as DNA carriers has high potential for gene therapy applications. Pectin is a structural plant polysaccharide heterogeneous with respect to its chemical structure. It contains branches rich in galactose residues which serve as potential ligands for membrane receptors interaction. In order to make the anionic pectin applicable for DNA complexation, it was modified with three different amine groups (cationic). Pectin-NH2 was prepared by modifying the galacturonic acids carboxyl groups with primary amine groups and further modified to generate pectin-T (T=N+H(CH3)(2)) and pectin-NH2-Q (Q=N+(CH3)(3)). All three modified pectins formed complexes with plasmid DNA as indicated by gel electrophoresis analysis. The size and morphology of pectin-NH2/DNA complexes were examined by transmission electron microscopy (TEM). Transfection experiments were carried out with human embryonic kidney cell lines (HEK293), using plasmid DNA encoding for green fluorescence protein (GFP). Transfection efficiency was analyzed by flow cytometry analysis, using FACS. Pectin-NH2-Q was the most efficient carrier. Addition of chloroquine ("lysosomotropic" agent) to transfection medium substantially enhanced the HEK293 transfection, indicating that endocytosis is the preferable internalization pathway and implies on the complex inability to escape the endosome. Pectin's galactose residues contribution to transfection was examined by inhibiting pectin binding to membrane receptors (galectins), using galactose and lactose as competitive inhibitors to this interaction. Resulting reduction of transfection efficiency demonstrated the importance of pectin's galactose residues to HEK293 transfection. Suggesting the modified pectin is a promising non-viral carrier for targeted gene delivery to cancer cells with galactose-binding lectins on their surface.

Smart polymers for responsive drug-delivery systems.

The rapid advancement of biomedical research has led to many creative applications for biocompatible polymers. As modern medicine discerns more mechanisms, both of physiology and of pathophysiology, the approach to healing is to mimic, or if possible, to recreate the physiology of healthy functioning. Thus, the area of smart polymers for responsive drug delivery has evolved. The developments fall under two categories: externally regulated or pulsatile systems (also known as 'open-loop' systems) and self-regulated systems (also known as 'closed-loop'). The externally controlled devices apply external triggers for pulsatile delivery such as: ultrasonic, magnetic, electric, light and chemical or biochemical agents. The self-regulated systems, on the other hand, are defined as systems where the controlled variable is detected, and as a result, the system output is adjusted accordingly. The release rate is controlled by feedback information, without any external intervention. The self-regulated systems utilize several approaches for the rate control mechanisms such as thermal, pH-sensitive polymers, enzyme-substrate reactions, pH-sensitive drug solubility, competitive binding, antibody interactions and metal-concentration-dependent hydrolysis.