Therapeutic value of homeoprotein signaling pathways.

Cell signaling based on homeoprotein transfer is a pathway with developmental and physiological functions. For a few transcription factors of this family, primarily ENGRAILED1, ENGRAILED2 and OTX2, their physiological functions have led to therapeutic strategies in animal models of human diseases, including Parkinson's disease, amyotrophic lateral sclerosis, amblyopia and anxiety-related disorders. In mesencephalic dopaminergic neurons which degenerate in Parkinson's disease, ENGRAILED1/2 have cell autonomous activities, but their transducing properties enables their use as therapeutic proteins. In contrast, in spinal alpha-motoneurons, which are lost in amyotrophic lateral sclerosis, ENGRAILED1 is supplied by V1 interneurons. Thus, its use as a therapeutic protein to protect alpha-motoneurons against degeneration mimics its normal non-cell autonomous neurotrophic activity. OTX2, synthesized and secreted by the choroid plexus, is transferred to parvalbumin interneurons and exerts regulatory functions controlling cerebral cortex plasticity. Understanding the latter OTX2 function has led to strategies for manipulating visual acuity and anxiety-like behavior in adult mice. In this review, we describe these cases and what is known about the involved molecular mechanisms. Because the transduction sequences are conserved in most of the few hundred homeoproteins, we argue how this family of molecules constitutes an important reservoir of physiological knowledge, with potential consequences in the search for new therapeutic strategies.

Protective effect of Tat fused HPCA protein on neuronal cell death caused by ischemic injury.

Background: Bain ischemia is a disease that occurs for various reasons, induces reactive oxygen species (ROS), and causes fatal damage to the nervous system. Protective effect of HPCA on ischemic injury has not been extensively studied despite its significance in regulating calcium homeostasis and promoting neuronal survival in CA1 region of the brain. Objective: We investigate the role of HPCA in ischemic injury using a cell-permeable Tat peptide fused HPCA protein (Tat-HPCA). Methods: Western blot analysis determined the penetration of Tat-HPCA into HT-22 cells and apoptotic signaling pathways. 5-CFDA, AM, DCF-DA, and TUNEL staining confirmed intracellular ROS production and DNA damage. The intracellular Ca2+ was measured in primary cultured neurons treated with H2O2. Protective effects were examined using immunohistochemistry and cognitive function tests by passive avoidance test and 8-arm radial maze test. Results: Tat-HPCA effectively penetrated into HT-22 cells and inhibited H2O2-induced apoptosis, oxidative stress, and DNA fragmentation. It also effectively inhibited phosphorylation of JNK and regulated the activation of Caspase, Bax, Bcl-2, and PARP, leading to inhibition of apoptosis. Moreover, Ca2+ concentration decreased in cells treated with Tat-HPCA in primary cultured neurons. In an animal model of ischemia, Tat-HPCA effectively penetrated the hippocampus, inhibited cell death, and regulated activities of astrocytes and microglia. Additionally, Cognitive function tests show that Tat-HPCA improves neurobehavioral outcomes after cerebral ischemic injury. Conclusion: These results suggest that Tat-HPCA might have potential as a therapeutic agent for treating oxidative stress-related diseases induced by ischemic injury, including ischemia.

Optimizing enzyme-responsive polymersomes for protein-based therapies.

Aims: Stimuli-responsive polymersomes are promising tools for protein-based therapies, but require deeper understanding and optimization of their pathology-responsive behavior. Materials & methods: Hyaluronic acid (HA)-poly(b-lactic acid) (PLA) polymersomes self-assembled from block copolymers of varying molecular weights of HA were compared for their physical properties, degradation and intracellular behavior. Results: Major results showed increasing enzyme-responsivity associated with decreasing molecular weight. The major formulation differences were as follows: the HA(5 kDa)-PLA formulation exhibited the most pronounced release of encapsulated proteins, while the HA(7 kDa)-PLA formulation showed the most different release behavior from neutral. Conclusion: We have discovered design rules for HA-PLA polymersomes for protein delivery, with lower molecular weight leading to higher encapsulation efficiency, greater release and greater intracellular uptake.

Recombinant protein delivery enables modulation of the phototransduction cascade in mouse retina.

Inherited retinal dystrophies are often associated with mutations in the genes involved in the phototransduction cascade in photoreceptors, a paradigmatic signaling pathway mediated by G protein-coupled receptors. Photoreceptor viability is strictly dependent on the levels of the second messengers cGMP and Ca2+. Here we explored the possibility of modulating the phototransduction cascade in mouse rods using direct or liposome-mediated administration of a recombinant protein crucial for regulating the interplay of the second messengers in photoreceptor outer segments. The effects of administration of the free and liposome-encapsulated human guanylate cyclase-activating protein 1 (GCAP1) were compared in biological systems of increasing complexity (in cyto, ex vivo, and in vivo). The analysis of protein biodistribution and the direct measurement of functional alteration in rod photoresponses show that the exogenous GCAP1 protein is fully incorporated into the mouse retina and photoreceptor outer segments. Furthermore, only in the presence of a point mutation associated with cone-rod dystrophy in humans p.(E111V), protein delivery induces a disease-like electrophysiological phenotype, consistent with constitutive activation of the retinal guanylate cyclase. Our study demonstrates that both direct and liposome-mediated protein delivery are powerful complementary tools for targeting signaling cascades in neuronal cells, which could be particularly important for the treatment of autosomal dominant genetic diseases.

Tat-CIAPIN1 Prevents Pancreatic ß-Cell Death in hIAPP-Induced RINm5F Cells and T2DM Animal Model.

It is well known that the cytokine-induced apoptosis inhibitor 1 (CIAPIN1) protein plays an important role in biological progresses as an anti-apoptotic protein. Human islet amyloid peptide (hIAPP), known as amylin, is caused to pancreatic ß-cell death in type 2 diabetes mellitus (T2DM). However, the function of CIAPIN1 protein on T2DM is not yet well studied. Therefore, we investigated the effects of CIAPIN1 protein on a hIAPP-induced RINm5F cell and T2DM animal model induced by a high-fat diet (HFD) and streptozotocin (STZ). The Tat-CIAPIN1 protein reduced the activation of mitogen-activated protein kinase (MAPK) and regulated the apoptosis-related protein expression levels including COX-2, iNOS, Bcl-2, Bax, and Caspase-3 in hIAPP-induced RINm5F cells. In a T2DM mice model, the Tat-CIAPIN1 protein ameliorated the pathological changes of pancreatic ß-cells and reduced the fasting blood glucose, body weight and hemoglobin Alc (HbAlc) levels. In conclusion, the Tat-CIAPIN1 protein showed protective effects against T2DM by protection of ß-cells via inhibition of hIAPP toxicity and by regulation of a MAPK signal pathway, suggesting CIAPIN1 protein can be a therapeutic protein drug candidate by beneficial regulation of T2DM.

Recent Progress of Rational Modified Nanocarriers for Cytosolic Protein Delivery.

Therapeutic proteins garnered significant attention in the field of disease treatment. In comparison to small molecule drugs, protein therapies offer distinct advantages, including high potency, specificity, low toxicity, and reduced carcinogenicity, even at minimal concentrations. However, the full potential of protein therapy is limited by inherent challenges such as large molecular size, delicate tertiary structure, and poor membrane penetration, resulting in inefficient intracellular delivery into target cells. To address these challenges and enhance the clinical applications of protein therapies, various protein-loaded nanocarriers with tailored modifications were developed, including liposomes, exosomes, polymeric nanoparticles, and nanomotors. Despite these advancements, many of these strategies encounter significant issues such as entrapment within endosomes, leading to low therapeutic efficiency. In this review, we extensively discussed diverse strategies for the rational design of nanocarriers, aiming to overcome these limitations. Additionally, we presented a forward-looking viewpoint on the innovative generation of delivery systems specifically tailored for protein-based therapies. Our intention was to offer theoretical and technical support for the development and enhancement of nanocarriers capable of facilitating cytosolic protein delivery.

Biotherapy of experimental acute kidney injury: emerging novel therapeutic strategies.

Acute kidney injury (AKI) is a complex and heterogeneous disease with high incidence and mortality, posing a serious threat to human life and health. Usually, in clinical practice, AKI is caused by crush injury, nephrotoxin exposure, ischemia-reperfusion injury, or sepsis. Therefore, most AKI models for pharmacological experimentation are based on this. The current research promises to develop new biological therapies, including antibody therapy, non-antibody protein therapy, cell therapy, and RNA therapy, that could help mitigate the development of AKI. These approaches can promote renal repair and improve systemic hemodynamics after renal injury by reducing oxidative stress, inflammatory response, organelles damage, and cell death, or activating cytoprotective mechanisms. However, no candidate drugs for AKI prevention or treatment have been successfully translated from bench to bedside. This article summarizes the latest progress in AKI biotherapy, focusing on potential clinical targets and novel treatment strategies that merit further investigation in future pre-clinical and clinical studies.

A Review of Current and Prospective Treatments for Channelopathies, with a Focus on Gene and Protein Therapy.

Reduced cell surface expression or the malfunctioning of ion channels gives rise to a group of disorders known as channelopathies. To treat the underlying cause, the delivery and/or expression of a functional ion channel into the cell membrane of the cell of interest is required. Unfortunately, for most channelopathies, current treatment options are only symptomatic and treatments that rectify the underlying damage are still lacking. Within this context, approaches that rely on gene and protein therapy are required. Gene therapy would allow the expression of a functional protein, provided that the cellular machinery in the diseased cell could correctly fold and traffic the protein to the cell membrane. Whereas protein therapy would allow the direct delivery of a functional protein, provided that the purification process does not affect protein function and a suitable delivery vehicle for targeted delivery is used. In this review, we provide an overview of channelopathies and available symptomatic treatments. The current state of gene therapy approaches mainly using viral vectors is discussed, which is followed by the role of nanomedicine in protein therapy and how nanomedicine could be exploited for the delivery of functional ion channels to diseased cells.

Protective effects of cell permeable Tat-PIM2 protein on oxidative stress induced dopaminergic neuronal cell death.

Background: Oxidative stress is considered as one of the main causes of Parkinson's disease (PD), however the exact etiology of PD is still unknown. Although it is known that Proviral Integration Moloney-2 (PIM2) promotes cell survival by its ability to inhibit formation of reactive oxygen species (ROS) in the brain, the precise functional role of PIM2 in PD has not been fully studied yet. Objective: We investigated the protective effect of PIM2 against apoptosis of dopaminergic neuronal cells caused by oxidative stress-induced ROS damage by using the cell permeable Tat-PIM2 fusion protein in vitro and in vivo. Methods: Transduction of Tat-PIM2 into SH-SY5Y cells and apoptotic signaling pathways were determined by Western blot analysis. Intracellular ROS production and DNA damage was confirmed by DCF-DA and TUNEL staining. Cell viability was determined by MTT assay. PD animal model was induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and protective effects were examined using immunohistochemistry. Results: Transduced Tat-PIM2 inhibited the apoptotic caspase signaling and reduced the production of ROS induced by 1-methyl-4-phenylpyridinium (MPP+) in SH-SY5Y cells. Furthermore, we confirmed that Tat-PIM2 transduced into the substantia nigra (SN) region through the blood-brain barrier and this protein protected the Tyrosine hydroxylase-positive cells by observation of immunohistostaining. Tat-PIM2 also regulated antioxidant biomolecules such as SOD1, catalase, 4-HNE, and 8-OHdG which reduce the formation of ROS in the MPTP-induced PD mouse model. Conclusion: These results indicated that Tat-PIM2 markedly inhibited the loss of dopaminergic neurons by reducing ROS damage, suggesting that Tat-PIM2 might be a suitable therapeutic agent for PD.

Tat-RAN attenuates brain ischemic injury in hippocampal HT-22 cells and ischemia animal model.

Oxidative stress plays a key role in the pathogenesis of neuronal injury, including ischemia. Ras-related nuclear protein (RAN), a member of the Ras superfamily, involves in a variety of biological roles, such as cell division, proliferation, and signal transduction. Although RAN reveals antioxidant effect, its precise neuroprotective mechanisms are still unclear. Therefore, we investigated the effects of RAN on HT-22 cell which were exposed to H2O2-induced oxidative stress and ischemia animal model by using the cell permeable Tat-RAN fusion protein. We showed that Tat-RAN transduced into HT-22 cells, and markedly inhibited cell death, DNA fragmentation, and reactive oxygen species (ROS) generation under oxidative stress. This fusion protein also controlled cellular signaling pathways, including mitogen-activated protein kinases (MAPKs), NF-κB, and apoptosis (Caspase-3, p53, Bax and Bcl-2). In the cerebral forebrain ischemia animal model, Tat-RAN significantly inhibited both neuronal cell death, and astrocyte and microglia activation. These results indicate that RAN significantly protects against hippocampal neuronal cell death, suggesting Tat-RAN will help to develop the therapies for neuronal brain diseases including ischemic injury.

Tat-GSTpi Inhibits Dopaminergic Cells against MPP+-Induced Cellular Damage via the Reduction of Oxidative Stress and MAPK Activation.

Glutathione S-transferase pi (GSTpi) is a member of the GST family and plays many critical roles in cellular processes, including anti-oxidative and signal transduction. However, the role of anti-oxidant enzyme GSTpi against dopaminergic neuronal cell death has not been fully investigated. In the present study, we investigated the roles of cell permeable Tat-GSTpi fusion protein in a SH-SY5Y cell and a Parkinson's disease (PD) mouse model. In the 1-methyl-4-phenylpyridinium (MPP+)-exposed cells, Tat-GSTpi protein decreased DNA damage and reactive oxygen species (ROS) generation. Furthermore, this fusion protein increased cell viability by regulating MAPKs, Bcl-2, and Bax signaling. In addition, Tat-GSTpi protein delivered into the substantia nigra (SN) of mice brains protected dopaminergic neuronal cell death in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD animal model. Our results indicate that the Tat-GSTpi protein inhibited cell death from MPP+- and MPTP-induced damage, suggesting that it plays a protective role during the loss of dopaminergic neurons in PD and that it could help to identify the mechanism responsible for neurodegenerative diseases, including PD.

Protective Effects of PEP-1-GSTA2 Protein in Hippocampal Neuronal Cell Damage Induced by Oxidative Stress.

Glutathione S-transferase alpha 2 (GSTA2), a member of the glutathione S-transferase family, plays the role of cellular detoxification against oxidative stress. Although oxidative stress is related to ischemic injury, the role of GSTA2 against ischemia has not been elucidated. Thus, we studied whether GSTA2 prevents ischemic injury by using the PEP-1-GSTA2 protein which has a cell-permeable protein transduction domain. We revealed that cell-permeable PEP-1-GSTA2 transduced into HT-22 cells and markedly protected cell death via the inhibition of reactive oxygen species (ROS) production and DNA damage induced by oxidative stress. Additionally, transduced PEP-1-GSTA2 promoted mitogen-activated protein kinase (MAPK), and nuclear factor-kappaB (NF-κB) activation. Furthermore, PEP-1-GSTA2 regulated Bcl-2, Bax, cleaved Caspase-3 and -9 expression protein levels. An in vivo ischemic animal model, PEP-1-GSTA2, markedly prevented the loss of hippocampal neurons and reduced the activation of microglia and astrocytes. These findings indicate that PEP-1-GSTA2 suppresses hippocampal cell death by regulating the MAPK and apoptotic signaling pathways. Therefore, we suggest that PEP-1-GSTA2 will help to develop the therapies for oxidative-stress-induced ischemic injury.

From bench to clinic: Emerging therapies for corneal scarring.

Corneal diseases are one of the leading causes of moderate-to-severe visual impairment and blindness worldwide, after glaucoma, cataract, and retinal disease in overall importance. Given its tendency to affect people at a younger age than other blinding conditions such as cataract and glaucoma, corneal scarring poses a huge burden both on the individuals and society. Furthermore, corneal scarring and fibrosis disproportionately affects people in poorer and remote areas, making it a significant ophthalmic public health problem. Traditional medical strategies, such as topical corticosteroids, are not effective in preventing fibrosis or scars. Corneal transplantation, the only effective sight-restoring treatment for corneal scars, is curbed by challenges including a severe shortage of tissue, graft rejection, secondary conditions, cultural barriers, the lack of well-trained surgeons, operating rooms, and well-equipped infrastructures. Thanks to tremendous research efforts, emerging therapeutic options including gene therapy, protein therapy, cell therapy and novel molecules are in development to prevent the progression of corneal scarring and compliment the surgical options currently available for treating established corneal scars in clinics. In this article, we summarise the most relevant preclinical and clinical studies on emerging therapies for corneal scarring in recent years, showing how these approaches may prevent scarring in its early development.

Protective effect of GK2 fused BLVRA protein against oxidative stress-induced dopaminergic neuronal cell damage.

It is well known that oxidative stress is highly associated with Parkinson's disease (PD), and biliverdin reductase A (BLVRA) is known to have antioxidant properties against oxidative stress. In this study, we developed a novel N-acetylgalactosamine kinase (GK2) protein transduction domain (PTD) derived from adenosine A2A and fused with BLVRA to determine whether the GK2-BLVRA fusion protein could protect dopaminergic neuronal cells (SH-SY5Y) from oxidative stress in vitro and in vivo using a PD animal model. GK2-BLVRA was transduced into various cells, including SH-SY5Y cells, without cytotoxic effects, and this fusion protein protected SH-SY5Y cells and reduced reactive oxygen species production and DNA damage after 1-methyl-4-phenylpyridinium (MPP+ ) exposure. GK2-BLVRA suppressed mitogen-activated protein kinase (MAPK) activation and modulated apoptosis-related protein (Bcl-2, Bax, cleaved Caspase-3 and -9) expression levels. In the PD animal model, GK2-BLVRA transduced into the substantia nigra crossed the blood-brain barrier and markedly reduced dopaminergic neuronal cell death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced animals. These results indicate that our novel PTD GK-2 is useful for the transduction of protein, and GK2-BLVRA exhibits a beneficial effect against dopaminergic neuronal cell death in vitro and in vivo, suggesting that BLVRA can be used as a therapeutic agent for PD.

Glucose-Responsive Injectable Thermogels via Dynamic-Covalent Cross-Linking of Pluronic Micelles.

Stimuli-responsive hydrogels are an area of active discovery for approaches to deliver therapeutics in response to disease-specific indicators. Glucose-responsive delivery of insulin is of particular interest in better managing diabetes. Accordingly, hydrogels have been explored as platforms that enable both a rate and dose of insulin release aligning with the real-time physiological disease state; materials often include glucose sensing by dynamic-covalent cross-linking between phenylboronic acids (PBAs) and diols, with competition from ambient glucose reducing cross-link density of the material and accelerating release of encapsulated insulin. Yet, these materials historically have challenges with insulin leakage, offer limited glucose-responsive release of the insulin payload, and require unreasonably high injection pressures for syringe administration. Here, a thermogel platform prepared from temperature-induced micelles formed into a network by PBA-Diol cross-linking is optimized using a formulation-centered approach to maximize glucose-responsive insulin delivery. Importantly, the dual-responsive nature of this platform enables a low-viscosity sol at ambient temperature for facile injection, solidifying into a stable viscoelastic hydrogel network once in the body. The final optimized formulation affords acceleration in insulin release in response to glucose and enables single dose blood glucose control in diabetic rodents when subjected to multiple glucose challenges.

Hydrogels for brain repair: application to Parkinson's disease.

INTRODUCTION: Parkinson's disease is the second most common neurodegenerative disease. Currently, there are no curative therapies, with only symptomatic treatment available. One of the principal reasons for the lack of treatments is the problem of delivering drugs to the brain, mainly due to the blood-brain barrier. Hydrogels are presented as ideal platforms for delivering treatments to the brain ranging from small molecules to cell replacement therapies. AREAS COVERED: The potential application of hydrogel-based therapies for Parkinson's disease is addressed. The desirable composition and mechanical properties of these therapies for brain application are discussed, alongside the preclinical research available with hydrogels in Parkinson's disease. Lastly, translational and manufacturing challenges are presented. EXPERT OPINION: Parkinson's disease urgently needs novel therapies to delay its progression and for advanced stages, at which conventional therapies fail to control motor symptoms. Neurotrophic factor-loaded hydrogels with stem cells offer one of the most promising therapies. This approach may increase the striatal dopamine content while protecting and promoting the differentiation of stem cells although the generation of synapses between engrafted and host cells remains an issue to overcome. Other challenges to consider are related to the route of administration of hydrogels and their large-scale production, required to accelerate their translation toward the clinic.

Expression and Purification of Human Granzyme B Fusion Protein to Induce Targeted Apoptosis in PSMA Positive Prostate Cancer Cells.

BACKGROUND: Granzyme B can induce apoptosis in target cells by direct and indirect activation of caspases and cleavage of central caspase substrates. Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein and its expression increases following prostate cancer progression. OBJECTIVE: In this study, we designed a fusion protein including mutant granzyme B, the influenza virus hemagglutinin HA-2 N-terminal, and PSMA ligand to construct GrB-HA-PSMA ligand fusion protein as a molecular agent for selective targeting of PSMA-positive (LNCaP) cells. METHODS: The DNA sequence of our designed structure was synthesized and cloned into a pET28a expression vector. The recombinant protein was expressed in E. coli origami bacteria and then purified. The expression of the recombinant protein was verified by SDS PAGE and ELISA method. Furthermore, ELISA and flow cytometry assays were utilized to investigate the efficiency of binding and permeability of the recombinant protein into the LNCaP cells. Finally, cell proliferation and apoptosis rate were evaluated by MTT assay and flow cytometry assay, respectively. HeLa and PC3 cell lines were used as controls. RESULTS: The results showed that GrB-HA-PSMA ligand fusion protein could specifically bind and internalize into the PSMA-positive cells. Furthermore, treatment of the cells with GrB-HA-PSMA ligand fusion protein resulted in increased apoptotic cell death and decreased proliferation of LNCaP cells. CONCLUSION: Our findings indicate the specificity of GrB-HA-PSMA ligand fusion protein for PSMA-positive cells and suggest that this fusion protein is a potential candidate for prostate cancer targeted therapy.

Functionalizing multi-component bioink with platelet-rich plasma for customized in-situ bilayer bioprinting for wound healing.

In-situ three-dimensional (3D) bioprinting has been emerging as a promising technology designed to rapidly seal cutaneous defects according to their contour. Improvements in the formulations of multi-component bioink are needed to support cytocompatible encapsulation and biological functions. Platelet-rich plasma (PRP), as a source of patient-specific autologous growth factors, exhibits capabilities in tissue repair and rejuvenation. This study aimed to prepare PRP-integrated alginate-gelatin (AG) composite hydrogel bioinks and evaluate the biological effects in vitro and in vivo. 3D bioprinted constructs embedded with dermal fibroblasts and epidermal stem cells were fabricated using extrusion strategy. The integration of PRP not only improved the cellular behavior of seeded cells, but regulate the tube formation of vascular endothelial cells and macrophage polarization in a paracrine manner, which obtained an optimal effect at an incorporation concentration of 5%. For in-situ bioprinting, PRP integration accelerated the high-quality wound closure, modulated the inflammation and initiated the angiogenesis compared with the AG bioink. In conclusion, we revealed the regenerative potential of PRP, readily available at the bedside, as an initial signaling provider in multi-component bioink development. Combined with in-situ printing technology, it is expected to accelerate the clinical translation of rapid individualized wound repair.

pH-responsive dynamically cross-linked nanogels with effective endo-lysosomal escape for synergetic cancer therapy based on intracellular co-delivery of photosensitizers and proteins.

Co-delivery of photosensitizers (PSs) and protein drugs represents great potentiality for enhancing the efficiency of synergistic cancer therapy. However, the intricate tumor-microenvironment and the lack of nanoplatforms to co-deliver both into cancer cells and activate their functions significantly hinder the clinical translation of this combined approach for cancer treatment. Herein, a chlorine e6 (Ce6)-functionalized and pH-responsive dynamically cross-linked nanogel (Ce6@NG) is fabricated by formation of benzoic imine linkages between Ce6-modified methoxy poly (ethyleneglycol)-block-poly (diethylenetriamine)-L-glutamate-Ce6 [MPEG-b-P(Deta)LG-Ce6] and terephthalaldehyde as cross-linkers for effective intracellular co-delivery of Ce6 and cytochrome c (CC), which could form a novel combination therapy system (CC/Ce6@NGs). The pH-sensitive benzoic imine bonds in the CC/Ce6@NGs endow them with excellent systemic stability under normal physiological environment while this nanosystem can be further cationized to enhance cell uptake in acidic extracellular environment. Upon cellular internalization, CC/Ce6@NGs can rapidly escape from the endo/lysosomal compartments and subsequently activate Ce6 to generate cytotoxic singlet oxygen upon laser irradiation and release of CC to induce programmed cell death by complete cleavage of benzoic imines at more acidic intracellular environments. Importantly, the catalase-like activity of CC can decompose H2O2 to produce O2 for hypoxia alleviation and improvement of the photodynamic therapy (PDT) of cancer. Moreover, this enhanced synergistic anticancer activity is confirmed both in vitro and in vivo. In view of the versatile chemical conjugation, this research offers a promising and smart nanosystem for intracellular co-delivery of PSs and therapeutic proteins.

CTLA4-Ig prevents development of neutralizing antibody formation after continuous treatment with human FVIII in HA rats.

INTRODUCTION: Immunogenicity causing development of anti-drug antibodies (ADAs) are major challenges in the treatment of haemophilia, as well as other diseases where proteins are used for treatment. Furthermore, it is a complication for preclinical testing of such therapies in animal models. AIM: To investigate if the immunosuppressive drug CTLA4 immunoglobulin (CTLA4-Ig) can induce tolerance in haemophilia A (HA) rats receiving recombinant human coagulation factor VIII (rhFVIII) treatment. METHODS: Two different prophylactic rhFVIII compounds were given intravenously to HA rats for 4 weeks. Both rhFVIII compounds were co-administered with commercially available CTLA4-Ig or human IgG subclass 4 (hIgG4) as control, and blood samples were collected. To functionally test if pharmacological efficacy was retained, rats were subjected to a bleeding experiment under anaesthesia at end of study. RESULTS: The mean inhibitory level after 4 weeks in rats receiving rhFVIII and hIgG4 was 85.7 BU for octocog alfa and 37.4 BU for rurioctocog alfa pegol. In contrast, co-administration with CTLA4-Ig during rhFVIII therapy prevented the formation of ADAs (both binding and inhibitory) in 14/14 rats receiving octocog alfa and in 7/7 rats receiving rurioctocog alfa pegol. Moreover, we were able to show that the pharmacological efficacy of rhFVIII was preserved. CONCLUSION: In a rat model with spontaneous bleeding, co-administration of CTLA4-Ig with rhFVIII prevented antibody formation. No FVIII antibodies were detected, demonstrating that CTLA4-Ig co-administration can be applicable as a method to prevent immunogenicity, when evaluating human proteins in preclinical systems permitting continuous pharmacokinetic and pharmacodynamic assessment.