Improving corneal permeability of dexamethasone using penetration enhancing agents: First step towards achieving topical drug delivery to the retina.

With an ever-increasing burden of vision loss caused by diseases of the posterior ocular segment, there is an unmet clinical need for non-invasive treatment strategies. Topical drug application using eye drops suffers from low to negligible bioavailability to the posterior segment as a result of static and dynamic defensive ocular barriers to penetration, while invasive delivery systems are expensive to administer and suffer potentially severe complications. As the cornea is the main anatomical barrier to uptake of topically applied drugs from the ocular surface, we present an approach to increase corneal permeability of a corticosteroid, dexamethasone sodium-phosphate (DSP), using a novel penetration enhancing agent (PEA). We synthesised a novel polyacetylene (pAc) polymer and compared its activity to two previously described cell penetrating peptide (CPP) based PEAs, TAT and penetratin, with respect to increasing transcorneal permeability of DSP in a rapid ex-vivo porcine corneal assay over 60 min. The transcorneal apparent permeability coefficients (Papp) for diffusion of pAc, and fluorescein isothiocyanate (FITC) conjugated TAT and penetratin were up to 5 times higher (p < 0.001), when compared to controls. When pAc was used in formulation with DSP, an almost 5-fold significant increase was observed in Papp of DSP across the cornea (p = 0.0130), a significant 6-fold increase with TAT (p = 0.0377), and almost 7-fold mean increase with penetratin (p = 0.9540). Furthermore, we investigated whether the PEAs caused any irreversible damage to the barrier integrity of the corneal epithelium by measuring transepithelial electrical resistance (TEER) and immunostaining of tight junction proteins using zonula occludens-1 (ZO-1) and occludin antibodies. There was no damage or structural toxicity, and the barrier integrity was preserved after PEA application. Finally, an in-vitro cytotoxicity assessment of all PEAs in human retinal pigment epithelium cells (ARPE-19) demonstrated that all PEAs were very well-tolerated, with IC50 values of 64.79 mM for pAc and 1335.45 µM and 87.26 µM for TAT and penetratin, respectively. Our results suggest that this drug delivery technology could potentially be used to achieve a significantly higher intraocular therapeutic bioavailability after topical eye drop administration, than currently afforded.

Tat-p27 Ameliorates Neuronal Damage Reducing α-Synuclein and Inflammatory Responses in Motor Neurons After Spinal Cord Ischemia.

p27Kip1 (p27) regulates the cell cycle by inhibiting G1 progression in cells. Several studies have shown conflicting results on the effects of p27 against cell death in various insults. In the present study, we examined the neuroprotective effects of p27 against H2O2-induced oxidative stress in NSC34 cells and against spinal cord ischemia-induced neuronal damage in rabbits. To promote delivery into NSC34 cells and motor neurons in the spinal cord, Tat-p27 fusion protein and its control protein (Control-p27) were synthesized with or without Tat peptide, respectively. Tat-p27, but not Control-27, was efficiently introduced into NSC34 cells in a concentration- and time-dependent manner, and the protein was detected in the cytoplasm. Tat-p27 showed neuroprotective effects against oxidative stress induced by H2O2 treatment and reduced the formation of reactive oxygen species, DNA fragmentation, and lipid peroxidation in NSC34 cells. Tat-p27, but not Control-p27, ameliorated ischemia-induced neurological deficits and cell damage in the rabbit spinal cord. In addition, Tat-p27 treatment reduced the expression of α-synuclein, activation of microglia, and release of pro-inflammatory cytokines such as interleukin-1ß and tumor necrosis factor-α in the spinal cord. Taken together, these results suggest that Tat-p27 inhibits neuronal damage by decreasing oxidative stress, α-synuclein expression, and inflammatory responses after ischemia.

Moderately Inducing Autophagy Reduces Tertiary Brain Injury after Perinatal Hypoxia-Ischemia.

Recent studies of cerebral hypoxia-ischemia (HI) have highlighted slowly progressive neurodegeneration whose mechanisms remain elusive, but if blocked, could considerably improve long-term neurological function. We previously established that the cytokine transforming growth factor (TGF)ß1 is highly elevated following HI and that delivering an antagonist for TGFß receptor activin-like kinase 5 (ALK5)-SB505124-three days after injury in a rat model of moderate pre-term HI significantly preserved the structural integrity of the thalamus and hippocampus as well as neurological functions associated with those brain structures. To elucidate the mechanism whereby ALK5 inhibition reduces cell death, we assessed levels of autophagy markers in neurons and found that SB505124 increased numbers of autophagosomes and levels of lipidated light chain 3 (LC3), a key protein known to mediate autophagy. However, those studies did not determine whether (1) SB was acting directly on the CNS and (2) whether directly inducing autophagy could decrease cell death and improve outcome. Here we show that administering an ALK5 antagonist three days after HI reduced actively apoptotic cells by ~90% when assessed one week after injury. Ex vivo studies using the lysosomal inhibitor chloroquine confirmed that SB505124 enhanced autophagy flux in the injured hemisphere, with a significant accumulation of the autophagic proteins LC3 and p62 in SB505124 + chloroquine treated brain slices. We independently activated autophagy using the stimulatory peptide Tat-Beclin1 to determine if enhanced autophagy is directly responsible for improved outcomes. Administering Tat-Beclin1 starting three days after injury preserved the structural integrity of the hippocampus and thalamus with improved sensorimotor function. These data support the conclusion that intervening at this phase of injury represents a window of opportunity where stimulating autophagy is beneficial.

Mn-TAT PTD-Ngb ameliorates inflammation through the elimination of damaged mitochondria and the activation of Nrf2-antioxidant signaling pathway.

Inflammation, mitochondrial dysfunction and oxidative stress are closely associated with neurological diseases. In this study, Mn-TAT PTD-Ngb, a novel artificial recombinant protein, exerted inhibitory effects on the inflammatory response and inflammasome activation. During the lipopolysaccharide (LPS)-induced inflammatory response, Mn-TAT PTD-Ngb suppressed the nuclear translocation of nuclear factor kappa B (NF-κB) and the release of proinflammatory cytokines and attenuated the phosphorylation of mitogen-activated protein kinase (MAPK). Furthermore, the recombinant protein blocked reactive oxygen species (ROS) production, abated mitochondrial dysfunction and significantly suppressed the assembly of the inflammasome, which led to the overproduction of proinflammatory cytokines IL-1ß and IL-18. Mn-TAT PTD-Ngb increased the level of nuclear factor-erythroid 2 -related factor 2 (Nrf2), which protected against oxidative stress and improved pyroptosis. Mn-TAT PTD-Ngb might be a promising drug for curing neurological diseases.

Supplementation of cryopreservation medium with TAT-Peroxiredoxin 2 fusion protein improves human sperm quality and function.

OBJECTIVE: To investigate the potential effects of TAT-PRDX2 protein supplementation to the cryopreservation medium on post-thaw sperm quality and function. DESIGN: In vitro prospective study. SETTING: Medical university hospital. PATIENT(S): Fifty normozoospermic, 50 asthenozoospermic, and 50 oligoasthenozoospermic men undergoing semen analysis for couple infertility. INTERVENTION(S): Each semen sample was divided into three aliquots: fresh, cryopreserved control (without additive), and cryopreserved with TAT-PRDX2 protein. MAIN OUTCOME MEASURE(S): Sperm motility, viability, mitochondrial potential, and DNA damage as well as reactive oxygen species (ROS) levels and lipid peroxidation were analyzed. Acrosome reaction and zona-free hamster oocyte penetration tests were performed to assess the fertilization ability of cryopreserved spermatozoa. RESULT(S): In normozoospermic and asthenozoospermic groups, the addition of 150 µg/mL TAT-PRDX2 significantly reduced intracellular ROS and malondialdehyde levels and enhanced post-thaw sperm motility and viability when compared with the cryopreserved control of the respective groups but did not produce any significant protective effect in the oligoasthenozoospermic group. Mitochondrial potential was significantly increased, whereas DNA fragmentation was significantly decreased, after TAT-PRDX2 supplementation only in the asthenozoospermic group when compared with the cryopreserved control. Although the penetration rate and the penetration index were not markedly improved, TAT-PRDX2 supplementation obviously reduced spontaneous acrosome reaction and increased calcium ionophore-induced acrosome reaction in the normozoospermic and asthenozoospermic groups. CONCLUSION(S): TAT-PRDX2 protein effectively exerted cryoprotective effects on spermatozoa by reducing intracellular ROS level and thereby improved post-thaw sperm quality and function, especially for asthenozoospermic samples. TAT-PRDX2 protein is a promising additive for developing a new and highly efficient semen cryoprotectant.

The effects of repetitive stress on tat protein-induced pro-inflammatory cytokine release and steroid receptor expression in the hippocampus of rats.

Human immunodeficiency virus type 1 (HIV-1) affects the central nervous system (CNS) that may lead to the development of HIV-associated neuropathologies. Tat protein is one of the viral proteins that have been linked to the neurotoxic effects of HIV. Since many individuals living with HIV often experience significant adverse circumstances, the present study investigated whether exposure to stressful conditions would exacerbate harmful effects of tat protein on brain function. Tat protein (10 µg/10 µl) was injected bilaterally into the dorsal hippocampus of the animal using stereotaxic techniques. The control group received an injection of saline (10 µl). Some control and tat protein-treated animals were subjected to restrain stress for 6 h per day for 28 days and compared to a non-stress group. All animals underwent two behavioural tests, the open field test (OFT) and the novel object recognition test (NORT) to assess their mood state and cognitive function respectively. The release of pro-inflammatory cytokines (TNF-α and IL-1ß) and the expression of mineralocorticoid (MR) and glucocorticoid (GR) receptors were also measured to see whether the impact of the repetitive stress on Tat protein-induced behavioural effects was mediated by elements of the immune system and the HPA axis. Rats treated with tat protein showed the following behavioural changes when compared to control animals: there was a significant decrease in time spent in the center of the open field during the OFT, a significant reduction in time spent with the novel object during the NORT, but no change in locomotor activity. Real-time PCR data showed that the expression levels of GR and MR mRNA were significantly reduced, while Western blot analysis showed that the protein expression levels of TNF-α and IL-1ß were significantly increased. The present findings indicated that injection of tat protein into the hippocampus of rats not subjected to stress may lead to anxiety-like behaviour and deficits in learning and memory. Tat-treated animals subjected to stress evoked only a modest effect on their behaviour and neurochemistry, while stress alone led to behavioural and neurochemical changes similar to tat protein.

Microfluidic self-assembly of a combinatorial library of single- and dual-ligand liposomes for in vitro and in vivo tumor targeting.

Precise engineering of nanoparticles with systematically varied properties (size, charge surface properties, targeting ligands, etc.) remains a challenge, limiting the effective optimization of nanoparticles for particular applications. Herein we report a single-step microfluidic combinatorial approach for producing a library of single and dual-ligand liposomes with systematically-varied properties including size, zeta potential, targeting ligand, ligand density, and ligand ratio. A targeting ligand folic acid and a cell penetrating peptide TAT were employed to achieve the optimal synergistic targeting effect. In 2D cell monolayer models, the single-ligand folic acid modified liposome didn't show any enhanced cellular uptake, while the incorporation of TAT peptide "switched on" the function of folic acid, and induced significant elevated cellular uptake compared to the single ligand modified liposomes, showing a strong synergistic targeting effect. The folic acid and TAT peptide dual-ligand liposome also demonstrated enhanced tumor penetration as observed using 3D tumor spheroid models. The in vivo study further confirmed the improved tumor targeting and longer tumor retention (up to 72 h) of the dual-ligand liposomes. Our work not only proved the versatility of this microfluidic combinatorial approach in producing libraries of multifunctional liposomes with controlled properties but also revealed the great potential of the optimized liposome formulation for synergistic targeting effects.

Intranasal Administration of TAT-Conjugated Lipid Nanocarriers Loading GDNF for Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder (ND), characterized by the loss of dopaminergic neurons, microglial activation, and neuroinflammation. Current available treatments in clinical practice cannot halt the progression of the disease. During the last few years, growth factors (GFs) have been raised as a promising therapeutic approach to address the underlying neurodegenerative process. Among others, glial cell-derived neurotrophic factor (GDNF) is a widely studied GF for PD. However, its clinical use is limited due to its short half life, rapid degradation rate, and difficulties in crossing the blood-brain barrier (BBB). Lately, intranasal administration has appeared as an alternative non-invasive way to bypass the BBB and target drugs directly to the central nervous system (CNS). Thus, the aim of this work was to develop a novel nanoformulation to enhance brain targeting in PD through nasal administration. For that purpose, GDNF was encapsulated into chitosan (CS)-coated nanostructured lipid carriers, with the surface modified with transactivator of transcription (TAT) peptide (CS-nanostructured lipid carrier (NLC)-TAT-GDNF). After the physiochemical characterization of nanoparticles, the in vivo study was performed by intranasal administration to a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. The CS-NLC-TAT-GDNF-treated group revealed motor recovery which was confirmed with immunohistochemistry studies, showing the highest number of tyrosine hydroxylase (TH+) fibers in the striatum and TH+ neuron levels in the substantia nigra. Moreover, ionizing calcium-binding adaptor molecule 1 immunohistochemistry was performed, revealing that CS-NLC-TAT-GDNF acts as a modulator on microglia activation, obtaining values similar to control. Therefore, it may be concluded that the intranasal administration of CS-NLC-TAT-GDNF may represent a promising therapy for PD treatment.

Tat-protein disulfide-isomerase A3: a possible candidate for preventing ischemic damage in the spinal cord.

In the present study, we searched for possible candidates that can prevent ischemic damage in the rabbit spinal cord. For this study, we used two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, in sham- and ischemia-operated animals. As the level of protein disulfide-isomerase A3 (PDIA3) significantly decreased 3 h after ischemia/reperfusion, we further investigated its possible role against ischemic damage using an in vitro spinal cord cell line and in vivo spinal cord ischemic model. The administration of Tat-PDIA3 significantly reduced the hydrogen peroxide-induced formation of reactive oxygen species and cell death, based on terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling and a colorimetric WST-1 assay. Further, Tat-PDIA3 significantly ameliorated the ischemia-induced deficits in motor function, based on Tarlov's criteria, 24-72 h after ischemia/reperfusion, as well as the degeneration of motor neurons in the ventral horn 72 h after ischemia/reperfusion. Tat-PDIA3 administration also reduced the ischemia-induced activation of microglia and lipid peroxidation in the motor neurons 72 h after ischemia/reperfusion. PDIA3 also potentially ameliorated the ischemia-induced increase in oxidative markers in serum and decreased the activity of Cu,Zn-superoxide dismutase, Mn-superoxide dismutase, and glutathione peroxidase in spinal cord homogenates, 24 h and 72 h after ischemia/reperfusion. These results suggest that Tat-PDIA3 could be used to protect spinal cord neurons from ischemic damage, due to its modulatory action on the oxidative/anti-oxidative balance. Tat-PDIA3 could be applicable to protects neurons from the ischemic damage induced by thoracoabdominal aorta obstruction.

Targeting brain tumors by intra-arterial delivery of cell-penetrating peptides: a novel approach for primary and metastatic brain malignancy.

Computational modeling shows that intra-arterial delivery is most efficient when the delivered drugs rapidly and avidly bind to the target site. The cell-penetrating peptide trans-activator of transcription (TAT) is a candidate carrier molecule that could mediate such specificity for brain tumor chemotherapeutics. To test this hypothesis we first performed in vitro studies testing the uptake of TAT by one primary and three potentially metastatic brain cancer cell lines (9L, 4T-1, LLC, SKOV-3). Then we performed in vivo studies in a rat model where TAT was delivered either intra-arterially (IA) or intravenously (IV) to 9L brain tumors. We observed robust uptake of TAT by all tumor cell lines in vitro. Flow cytometry and confocal microscopy revealed a rapid uptake of fluorescein-labeled TAT within 5 min of exposure to the cancer cells. IA injections done under transient cerebral hypoperfusion (TCH) generated a four-fold greater tumor TAT concentration compared to conventional IV injections. We conclude that it is feasible to selectively target brain tumors with TAT-linked chemotherapy by the IA-TCH method.

Enhanced sublingual immunotherapy by TAT-fused recombinant allergen in a murine rhinitis model.

Allergen-specific sublingual immunotherapy (SLIT) is well known as an effective and non-invasive route to induce allergy desensitization. The goal of this study was to investigate whether a TAT-fused recombinant allergen could enhance SLIT efficacy. BALB/c mice sensitized to the main allergen (Che a 3) of Chenopodium album pollen were treated sublingually either with rChe a 3 (100µg/dose) or rTAT-Che a 3 (100µg/dose), two times per week for eight weeks. SLIT with rTAT-Che a 3 led to significantly greater allergen-specific IgG2a than rChe a 3; however, neither rTAT-Che a 3 nor rChe a 3 affected allergen-specific IgE or IgG1 antibody levels. In addition, interleukin 4 (IL-4) levels in re-stimulated splenocytes from the rTAT-Che a 3 mice were significantly lower than in those from the rChe a 3 mice, while interferon-γ (IFN-γ) was significantly greater in the rChe a 3 mice than in the rTAT-Che a 3 mice. Furthermore, sublingual administration of rTAT-Che a 3 induced significantly greater TGF-ß secretion in re-stimulated splenocytes than administration of rChe a 3. Accordingly, SLIT with rTAT-Che a 3 led to significantly greater expression of TGF-ß- and Foxp3-specific mRNAs in the splenocytes than in those from the rChe a 3 mice. Our findings demonstrate that TAT-fused rChe a 3 suppressed the allergic response through preferential enhancement of systemic regulatory T-cell (Treg)-mediated immunity responses, likely by facilitating allergen capture and presentation by sublingual Langerhans-like dendritic cells.

Human-derived fusogenic peptides for the intracellular delivery of proteins.

The cytosolic delivery of therapeutic proteins (e.g., antibodies or enzymes) by cell-penetrating peptides (CPPs), such as a human immunodeficiency virus-derived TAT peptide, is facilitated by fusogenic peptides (FPs). For instance, we recently demonstrated that an FP, B18, which is derived from a sea urchin gamete fusion protein, promotes endosomal escape of an enhanced green fluorescent protein (eGFP)-TAT fusion protein directly conjugated to it. However, the potential clinical use of FPs raises concerns because all conventional FPs are non-human-derived. To solve this problem, we have attempted to identify novel human-derived FPs from two human proteins, including a human sperm protein, IZUMO1, which is involved in gamete recognition and fusion, and a human endogenous retroviral envelope protein, Syncytin1, which is involved in placental morphogenesis. Partial peptides from the core domains of the abovementioned proteins were chosen as candidates to generate human-derived FPs. We prepared fusion proteins of these peptides with eGFP and TAT in Escherichia coli and observed the localization of these fusion proteins in HeLa cells using confocal microscopy. Our results suggested that a 19-residues peptide of Syncytin1 (positions 322-340), named S19, possessed strong intracellular uptake activities with no detectable cytotoxicity. In addition, we estimated the number of molecules that escaped from endosomes using a nuclear localization signal, suggesting that the S19 peptide stimulated the intracellular delivery of TAT-fused eGFP by ~90-fold. Furthermore, we confirmed that S19 promoted the intracellular delivery of eGFP to various human cell lines, including HeLa, A431, HepG2, and SK-N-SH. In addition, we demonstrated that not only eGFP but also SNAP-tag and ß-galactosidase were delivered efficiently and retained their activities.

Targeted iron oxide nanoparticles for the enhancement of radiation therapy.

To increase the efficacy of radiation, iron oxide nanoparticles can be utilized for their ability to produce reactive oxygen species (ROS). Radiation therapy promotes leakage of electrons from the electron transport chain and leads to an increase in mitochondrial production of the superoxide anion which is converted to hydrogen peroxide by superoxide dismutase. Iron oxide nanoparticles can then catalyze the reaction from hydrogen peroxide to the highly reactive hydroxyl radical. Therefore, the overall aim of this project was to utilize iron oxide nanoparticles conjugated to a cell penetrating peptide, TAT, to escape lysosomal encapsulation after internalization by cancer cells and catalyze hydroxyl radical formation. It was determined that TAT functionalized iron oxide nanoparticles and uncoated iron oxide nanoparticles resulted in permeabilization of the lysosomal membranes. Additionally, mitochondrial integrity was compromised when A549 cells were treated with both TAT-functionalized nanoparticles and radiation. Pre-treatment with TAT-functionalized nanoparticles also significantly increased the ROS generation associated with radiation. A long term viability study showed that TAT-functionalized nanoparticles combined with radiation resulted in a synergistic combination treatment. This is likely due to the TAT-functionalized nanoparticles sensitizing the cells to subsequent radiation therapy, because the nanoparticles alone did not result in significant toxicities.

Hypoglycemic activity and stability enhancement of human insulin-tat mixture loaded in elastic anionic niosomes.

This study aimed to investigate the synergistic effect of trans-activator of transcription (Tat) and niosomes for the improvement of hypoglycemic activity of orally delivered human insulin. The elastic anionic niosomes composing of Tween 61/cholesterol/dicetyl phosphate/sodium cholate at 1:1:0.05:0.02 molar ratio loaded with insulin-Tat mixture (1:3 molar ratio) was prepared. Deformability of the elastic anionic niosomes decreased after loaded with the mixture of 1.35 times. For the in vitro release, the insulin (T10 = 4 h) loaded in the elastic anionic niosomes indicated the slower release rate than insulin in the mixture (T10 = 3 h) loaded in niosomes. At room temperature (30 ± 2 °C), the mixture loaded in elastic anionic niosomes was more chemical stable than the free mixture of 1.3, 1.4 and 1.7 times after stored for 4, 8 and 12 weeks, respectively. Oral administration in the alloxan-induced diabetic mice of the mixture loaded in elastic anionic niosomes with the insulin doses at 25, 50 and 100 IU/kg body weight indicated significant hypoglycemic activity with the percentage fasting blood glucose reduction of 1.95, 2.10 and 2.10 folds of the subcutaneous insulin injection at 12 h, respectively. This study has demonstrated the synergistic benefits of Tat and elastic anionic niosomes for improving the hypoglycemic activity of the orally delivered human insulin as well as the stability enhancement of human insulin when stored at high temperature. The results from this study can be further developed as an effective oral insulin delivery.

Intranasal TAT-haFGF Improves Cognition and Amyloid-ß Pathology in an AßPP/PS1 Mouse Model of Alzheimer's Disease.

Neurotoxic amyloid-ß (Aß) peptide causing cognitive function disabilities is one of the most characteristic pathological features in Alzheimer's disease (AD). A novel fusion protein, TAT-haFGF, was administrated to AßPP/PS1 transgenic mice by intravenous (IV) injection and intranasal (IN) delivery, respectively, for 5 weeks to compare the pharmacodynamics between the two routes of administration. Our results showed that IN administration of TAT-haFGF improved cognition and reduced Aß plaques more significantly in AßPP/PS1 mice, when compared with IV injection. Our new findings suggest that TAT-haFGF might be a promising new therapy to attenuate AD pathological process.

Intraperitoneal Administration of a Novel TAT-BDNF Peptide Ameliorates Cognitive Impairments via Modulating Multiple Pathways in Two Alzheimer's Rodent Models.

Although Alzheimer's disease (AD) has been reported for more than 100 years, there is still a lack of effective cures for this devastating disorder. Among the various obstacles that hold back drug development, the blood-brain barrier (BBB) is one of them. Here, we constructed a novel fusion peptide by linking the active domain of brain-derived neurotrophic factor (BDNF) with an HIV-encoded transactivator of transcription (TAT) that has a strong membrane-penetrating property. After intraperitoneal injection, the eGFP-TAT could be robustly detected in different brain regions. By using scopolamine-induced rats and APPswe mice representing AD-like cholinergic deficits and amyloidosis, respectively, we found that intraperitoneal administration of the peptide significantly improved spatial memory with activation of the TrkB/ERK1/2/Akt pathway and restoration of several memory-associated proteins in both models. Administration of the peptide also modulated ß-amyloid and tau pathologies in APPswe mice, and it increased the amount of M receptor with modulation of acetylcholinesterase in scopolamine-induced rats. We conclude that intraperitoneal administration of our TAT-BDNF peptide could efficiently target multiple molecular pathways in the brain and improve the cognitive functions in AD-like rodent models.

Effects of low doses of Tat-PIM2 protein against hippocampal neuronal cell survival.

Oxidative stress is considered a major factor in various neuronal diseases including ischemia-reperfusion injury. Proviral Integration Moloney 2 (PIM2) proteins, one of the families of PIM kinases, play crucial roles in cell survival. However, the functions of PIM2 protein against ischemia are not understood. Therefore, the protective effects of PIM2 against oxidative stress-induced hippocampal HT22 cell death and brain ischemic injury were evaluated using Tat-PIM2, a cell permeable fusion protein. Tat-PIM2 protein transduced into hippocampal HT22 cells. Low doses of transduced Tat-PIM2 protein protected against oxidative stress-induced cell death including DNA damage and markedly inhibited the activation of mitogen activated protein kinase (MAPKs), NF-κB and the expression levels of Bax protein. Furthermore, Tat-PIM2 protein transduced into the CA1 region of the hippocampus and significantly prevented neuronal cell death in an ischemic insult animal model. These results indicated that low doses of Tat-PIM2 protein protects against oxidative stress-induced neuronal cell death, suggesting low doses of Tat-PIM2 protein provides a potential therapeutic agent against oxidative stress-induced neuronal diseases including ischemia.

Enhancing cancer targeting and anticancer activity by a stimulus-sensitive multifunctional polymer-drug conjugate.

Undesirable physicochemical properties, low tumor targeting, insufficient cell internalization, acquired drug resistance, and severe side effects significantly limit the applications of anticancer drugs. In this study, to improve the tumor targeting and drug efficacy of the poorly water-soluble drug, doxorubicin (DOX), a novel drug delivery platform (PEG-ppTAT-DOX) was developed, which contained a polyethylene glycol (PEG), a matrix metalloproteinase 2 (MMP2)-sensitive peptide linker (pp), a cell penetrating peptide (TAT), and a model drug (doxorubicin). The prepared drug platform possessed several key features, including: (i) the nanoparticle formation via the self-assembly; (ii) prevention of the non-specific interaction via the PEGylation; (iii) tumor targeting via the MMP2-mediated PEG deshielding and exposure of the TAT; (iv) the TAT-mediated cell internalization; (v) the TAT-induced endosomal escape; (vi) the inhibition of P-glycoprotein mediated drug efflux; and (vii) the TAT-medicated nuclear translocation. These cooperative functions ensured the improved tumor targetability, enhanced tumor cell internalization, improved intracellular distribution, and potentiated anticancer activity. Compared to the multi-component nanocarriers, the proposed simple but multifunctional polymer-drug conjugate might have greater potential for tumor-targeted drug delivery and enhanced chemotherapy.

Tat/HA2 Peptides Conjugated AuNR@pNIPAAm as a Photosensitizer Carrier for Near Infrared Triggered Photodynamic Therapy.

To achieve an efficiency of intracellular photosensitizers (PSs) delivery and efficacy of photodynamic therapy, we have developed a novel class of PS formulation for encapsulating sulfonated aluminum phthalocyanine (AlPcS4) by taking advantage of the membrane-disruptive peptides Tat/HA2 and the photothermally triggered delivery system using AuNR@pNIPAAm. The coordinated effects of cell penetrating peptide Tat and fusogenic peptide HA2 could enhance the efficient cellular internalization and endo/lysosome escape of PSs delivery systems. Singlet oxygen generation was inhibited due to the reaction between loaded AlPcS4 and Au nanorods, which indicated that the AlPcS4-loaded, AuNR@pNIPAAm delivery system might be nonphototoxic in the circulatory system. However, this PSs-loaded nanosystem became highly phototoxic as it underwent the near-infrared irradiation by using the combined lights of 808 and 680 nm. Upon irradiation, the Tat/HA2 conjugated AuNR@pNIPAAm-Pc elicited an active photodynamic response against the cancer cells, leading to effective cells killing via mitochondria-associated apoptotic pathway. This study also demonstrated improved PDT therapeutic efficacy after intravenous administration of Tat/HA2-AuNR@pNIPAAm-Pc and the subsequent lights irradiations in tumor-bearing mice. We describe here a strategy for enhanced photodynamic eradication of solid tumors by endo/lysosomal escape and highlight the great promise of peptide-based nanocarriers used for cancer therapy.

Transactivator of transcription (TAT) peptide- chitosan functionalized multiwalled carbon nanotubes as a potential drug delivery vehicle for cancer therapy.

Carbon nanotube (CNT)-based drug delivery vehicles might find great potential in cancer therapy via the combination of chemotherapy with photothermal therapy due to the strong optical absorbance of CNTs in the near-infrared region. However, the application of CNTs in cancer therapy was considerably constrained by their lack of solubility in aqueous medium, as well as the cytotoxicity caused by their hydrophobic surface. Intracellular delivery efficiency is another factor determining the application potential of CNTs in cancer therapy. In the present study, low-molecular-weight chitosan conjugated with transactivator of transcription (TAT) peptide was used for noncovalent functionalization of multiwalled carbon nanotubes (MWCNTs), aiming at providing a more efficient drug delivery vehicle for cancer therapy. The TAT-chitosan-conjugated MWCNTs (MWCNTs-TC) were further investigated for their water solubility, cytotoxicity, cell-penetrating capability, and accumulation in tumor. It was found that MWCNTs-TC were essentially nontoxic with satisfying water solubility, and they were more efficient in terms of cancer-targeted intracellular transport both in vitro and in vivo as compared with chitosan-modified MWCNTs (MWCNTs-CS), suggesting the great application potential of MWCNTs-TC in cancer therapy.