Peptide-guided delivery improves the therapeutic efficacy and safety of glucocorticoid drugs for treating acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening conditions with excessive inflammation in the lung. Glucocorticoids had been widely used for ALI/ARDS, but their clinical benefit remains unclear. Here, we tackled the problem by conjugating prednisolone (PSL) with a targeting peptide termed CRV. Systemically administered CRV selectively homes to the inflamed lung of a murine ALI model, but not healthy organs or the lung of healthy mice. The expression of the CRV receptor, retinoid X receptor ß, was elevated in the lung of ALI mice and patients with interstitial lung diseases, which may be the basis of CRV targeting. We then covalently conjugated PSL and CRV with a reactive oxygen species (ROS)-responsive linker in the middle. While being intact in blood, the ROS linker was cleaved intracellularly to release PSL for action. In vitro, CRV-PSL showed an anti-inflammatory effect similar to that of PSL. In vivo, CRV conjugation increased the amount of PSL in the inflamed lung but reduced its accumulation in healthy organs. Accordingly, CRV-PSL significantly reduced lung injury and immune-related side effects elsewhere. Taken together, our peptide-based strategy for targeted delivery of glucocorticoids for ALI may have great potential for clinical translation.

Co-administration of Transportan Peptide Enhances the Cellular Entry of Liposomes in the Bystander Manner Both In Vitro and In Vivo.

Liposomes have been widely used as a drug delivery vector. One way to further improve its therapeutic efficacy is to increase the cell entry efficiency. Covalent conjugation with cell-penetrating peptides (CPPs) and other types of ligands has been the mainstream strategy to tackle this issue. Although efficient, it requires additional chemical modifications on liposomes, which is undesirable for clinical translation. Our previous study showed that the transportan (TP) peptide, an amphiphilic CPP, was able to increase the cellular uptake of co-administered, but not covalently coupled, metallic nanoparticles (NPs). Termed bystander uptake, this process represents a simpler method to increase the cell entry of NPs without chemical modifications. Here, we extended our efforts to liposomes. Our results showed that co-administration with the TP peptide improved the internalization of liposome into a variety of cell lines in vitro. This effect was also observed in primary cells, ex vivo tumor slices, and in vivo tumor tissues. On the other hand, this peptide-assisted liposome internalization did not apply to cationic CPPs, which were the main inducers for bystander uptake in previous studies. We also found that TP-assisted bystander uptake of liposome is receptor dependent, and its activity is more sensitive to the inhibitors of the macropinocytosis pathway, underlining the potential cell entry mechanism. Overall, our study provides a simple strategy based on TP co-administration to increase the cell entry of liposomes, which may open up new avenues to apply TP peptides in nanotherapeutics.

iRGD-liposomes enhance tumor delivery and therapeutic efficacy of antisense oligonucleotide drugs against primary prostate cancer and bone metastasis.

Nucleotide-based drugs, such as antisense oligonucleotides (ASOs), have unique advantages in treating human diseases as they provide virtually unlimited ability to target any gene. However, their clinical translation faces many challenges, one of which is poor delivery to the target tissue in vivo. This problem is particularly evident in solid tumors. Here, we functionalized liposomes with a tumor-homing and -penetrating peptide, iRGD, as a carrier of an ASO against androgen receptor (AR) for prostate cancer treatment. The iRGD-liposomes exhibited a high loading efficiency of AR-ASO, and an efficient knockdown of AR gene products was achieved in vitro, including AR splice variants. In vivo, iRGD-liposomes significantly increased AR-ASO accumulation in the tumor tissue and decreased AR expression relative to free ASOs in prostate tumors established as subcutaneous xenografts. Similar results were obtained with intra-tibial xenografts modeling metastasis to bones, the predominant site of metastasis for prostate cancer. In treatment studies, iRGD-liposomes markedly improved the AR-ASO efficacy in suppressing the growth of both subcutaneous xenografts and intra-tibial xenografts. The inhibitory effect on tumor growth was also significantly prolonged by the delivery of the AR-ASO in the iRGD-liposomes. Meanwhile, iRGD-liposomes did not increase ASO accumulation or toxicity in healthy organs. Overall, we provide here a delivery system that can significantly increase ASO accumulation and efficacy in solid tumors. These benefits are achieved without significant side effects, providing a way to increase the antitumor efficacy of ASOs.

NG2 Proteoglycan Ablation Reduces Foam Cell Formation and Atherogenesis via Decreased Low-Density Lipoprotein Retention by Synthetic Smooth Muscle Cells.

OBJECTIVES: Obesity and hyperlipidemia are critical risk factors for atherosclerosis. Because ablation of NG2 proteoglycan in mice leads to hyperlipidemia and obesity, we investigated the impact of NG2 ablation on atherosclerosis in apoE null mice. APPROACH AND RESULTS: Immunostaining indicates that NG2 expression in plaque, primarily by synthetic smooth muscle cells, increases during atherogenesis. NG2 ablation unexpectedly results in decreased (30%) plaque development, despite aggravated obesity and hyperlipidemia. Mechanistic studies reveal that NG2-positive plaque synthetic smooth muscle cells in culture can sequester low-density lipoprotein to enhance foam-cell formation, processes in which NG2 itself plays direct roles. In agreement with these observations, low-density lipoprotein retention and lipid accumulation in the NG2/ApoE knockout aorta is 30% less than that seen in the control aorta. CONCLUSIONS: These results indicate that synthetic smooth muscle cell-dependent low-density lipoprotein retention and foam cell formation outweigh obesity and hyperlipidemia in promoting mouse atherogenesis. Our study sheds new light on the role of synthetic smooth muscle cells during atherogenesis. Blocking plaque NG2 or altering synthetic smooth muscle cells function may be promising therapeutic strategies for atherosclerosis.

Etchable plasmonic nanoparticle probes to image and quantify cellular internalization.

There is considerable interest in using nanoparticles as labels or to deliver drugs and other bioactive compounds to cells in vitro and in vivo. Fluorescent imaging, commonly used to study internalization and subcellular localization of nanoparticles, does not allow unequivocal distinction between cell surface-bound and internalized particles, as there is no methodology to turn particles 'off'. We have developed a simple technique to rapidly remove silver nanoparticles outside living cells, leaving only the internalized pool for imaging or quantification. The silver nanoparticle (AgNP) etching is based on the sensitivity of Ag to a hexacyanoferrate-thiosulphate redox-based destain solution. In demonstration of the technique we present a class of multicoloured plasmonic nanoprobes comprising dye-labelled AgNPs that are exceptionally bright and photostable, carry peptides as model targeting ligands, can be etched rapidly and with minimal toxicity in mice, and that show tumour uptake in vivo.

Evolution of double positive autoregulatory feedback loops in CYCLOIDEA2 clade genes is associated with the origin of floral zygomorphy.

Members of the CYCLOIDEA2 (CYC2) clade of the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF transcription factor genes are widely involved in controlling floral zygomorphy, a key innovation in angiosperm evolution, depending on their persistently asymmetric expression in the corresponding floral domains. However, it is unclear how this asymmetric expression is maintained throughout floral development. Selecting Primulina heterotricha as a model, we examined the expression and function of two CYC2 genes, CYC1C and CYC1D. We analyzed the role of their promoters in protein-DNA interactions and transcription activation using electrophoresis mobility shift assays, chromatin immunoprecipitation, and transient gene expression assays. We find that CYC1C and CYC1D positively autoregulate themselves and cross-regulate each other. Our results reveal a double positive autoregulatory feedback loop, evolved for a pair of CYC2 genes to maintain their expression in developing flowers. Further comparative genome analyses, together with the available expression and function data of CYC2 genes in the core eudicots, suggest that this mechanism might have led to the independent origins of floral zygomorphy, which are associated with plant-insect coevolution and the adaptive radiation of angiosperms.

Peptide targeting improves the delivery and therapeutic index of glucocorticoids to treat rheumatoid arthritis.

Rheumatoid arthritis (RA) is a prevalent autoimmune disease characterized by excessive inflammation in the joints. Glucocorticoid drugs are used clinically to manage RA symptoms, while their dosage and duration need to be tightly controlled due to severe adverse effects. Using dexamethasone (DEX) as a model drug, we explored here whether peptide-guided delivery could increase the safety and therapeutic index of glucocorticoids for RA treatment. Using multiple murine RA models such as collagen-induced arthritis (CIA), we found that CRV, a macrophage-targeting peptide, can selectively home to the inflammatory synovium of RA joints upon intravenous injection. The expression of the CRV receptor, retinoid X receptor beta (RXRB), was also elevated in the inflammatory synovium, likely being the basis of CRV targeting. CRV-conjugated DEX increased the accumulation of DEX in the inflamed synovium but not in healthy organs of CIA mice. Therefore, CRV-DEX demonstrated a stronger efficacy to suppress synovial inflammation and alleviate cartilage/bone destruction. Meanwhile, CRV conjugation reduced immune-related adverse effects of DEX even after a long-term use. Last, we found that RXRB expression was significantly elevated in human patient samples, demonstrating the potential of clinical translation. Taken together, we provide a novel, peptide-targeted strategy to improve the therapeutic efficacy and safety of glucocorticoids for RA treatment.

Virion stiffness regulates immature HIV-1 entry.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) undergoes a protease-mediated maturation process that is required for its infectivity. Little is known about how the physical properties of viral particles change during maturation and how these changes affect the viral lifecycle. Using Atomic Force Microscopy (AFM), we previously discovered that HIV undergoes a "stiffness switch", a dramatic reduction in particle stiffness during maturation that is mediated by the viral Envelope (Env) protein. RESULTS: In this study, we show that transmembrane-anchored Env cytoplasmic tail (CT) domain is sufficient to regulate the particle stiffness of immature HIV-1. Using this construct expressed in trans with viral Env lacking the CT domain, we show that increasing particle stiffness reduces viral entry activity in immature virions. A similar effect was also observed for immature HIV-1 pseudovirions containing Env from vesicular stomatitis virus. CONCLUSIONS: This linkage between particle stiffness and viral entry activity illustrates a novel level of regulation for viral replication, providing the first evidence for a biological role of virion physical properties and suggesting a new inhibitory strategy.

Identification of an ALK-2 inhibitor as an agonist for intercellular exchange and tumor delivery of nanomaterial.

Inefficient extravasation and penetration in solid tissues hinder the clinical outcome of nanoparticles (NPs). Recent studies have shown that the extravasation and penetration of NPs in solid tumor was mostly achieved via an active transcellular route. For this transport process, numerous efforts have been devoted to elucidate the endocytosis and subcellular trafficking of NPs. However, how they exit from one cell and re-enter into neighboring ones (termed intercellular exchange) remains poorly understood. We previously developed cellular assays that exclusively quantify the intercellular exchange of NPs in vitro. Our study showed that a significant portion of NPs are transferred inside extracellular vesicles (EVs). Pharmacological inhibition of EV biogenesis significantly reduced the tumor accumulation and vascular penetration of both inorganic and organic NPs in vivo. Intrigued by this result, we performed here a manual chemical screen with our assay, which identified that LDN-214117 (an inhibitor for activin receptor-like kinase-2, ALK-2) is an agonist of NP intercellular exchange. We further showed that LDN-214117 regulates the intercellular exchange by increasing the EV biogenesis. Mechanistic investigation showed that LDN-214117 functions via BMP (bone morphogenetic protein)-MAPK (mitogen-activated protein kinase) signaling pathway to increase EV biogenesis. We further demonstrated that LDN-214117 treatment in vivo enhanced the tumor accumulation and vascular penetration of a variety of NPs in multiple tumor models, which improves their antitumor efficacy. Overall, we showcase here the identification of a novel chemical compound with our intercellular exchange assays to modulate EV biogenesis and EV-mediated transport, thus boosting up the delivery and therapeutic efficacy of nanomaterial.

Extracellular Vesicles Mediate the Intercellular Exchange of Nanoparticles.

To exert their therapeutic effects, nanoparticles (NPs) often need to travel into the tissues composed of multilayered cells. Accumulative evidence has revealed the crucial role of transcellular transport route (entry into one cell, exocytosis, and re-entry into another) in this process. While NP endocytosis and subcellular transport are intensively characterized, the exocytosis and re-entry steps are poorly understood, which becomes a barrier for NP delivery into complex tissues. Here, the authors term the exocytosis and re-entry steps together as intercellular exchange. A collagen-based three-dimension assay is developed to specifically quantify the intercellular exchange of NPs, and distinguish the contributions of several potential mechanisms. The authors show that NPs can be exocytosed freely or enclosed inside extracellular vesicles (EVs) for re-entry, while direct cell-cell contact is hardly involved. EVs account for a significant fraction of NP intercellular exchange, and its importance in NP transport is demonstrated in vitro and in vivo. While freely released NPs engage with the same receptors for re-entry, EV-enclosed ones bypass this dependence. These studies provide an easy and precise system to investigate the intercellular exchange stage of NP delivery, and shed the first light in the importance of EVs in NP transport between cells and into complex tissues.

Synergistic Entry of Individual Nanoparticles into Mammalian Cells Driven by Free Energy Decline and Regulated by Their Sizes.

Cell entry is one of the common prerequisites for nanomaterial applications. Despite extensive studies on a homogeneous group of nanoparticles (NPs), fewer studies have been performed when two or more types of NPs were coadministrated. We previously described a synergistic cell entry process for two heterogeneous groups of NPs, where NPs functionalized with TAT (transactivator of transcription) peptide (T-NPs) stimulate the cellular uptake of coadministered unfunctionalized NPs (bystander NPs, B-NPs). Here, we show that the synergistic cell entry of NPs is driven by free energy decline and depends on B-NP sizes. Simulations showed that when separately placed initially, two NPs first move toward each other instead of initiating cell entry individually. Only T-NP invokes an inward bending of membrane mimicking endocytosis, which attracts the nearby NPs into the same "vesicle". A two-phase free energy decline of the entire system occurred as two NPs get closer until contact, which is likely the thermodynamic driver for synergistic NP coentry. Experimentally, we found that T-NPs increase the apparent affinity of B-NPs to plasma membrane, suggesting that T-NPs help B-NPs "trapped" in the endocytic vesicles. Next, we varied the sizes of B-NPs and found that bystander activity peaks around 50 nm. Simulations also showed that the size of B-NPs influences the free energy decline, and thus the tendency and dynamics of NP coentry. These efforts provide a system to further understand the synergistic cell entry among individual NPs or multiple NP types on a biophysical basis and shed light on the future design of nanostructures for intracellular delivery.

Macropinocytosis as a cell entry route for peptide-functionalized and bystander nanoparticles.

Endocytic pathways provide the primary route for therapeutic and diagnostic nanoparticles (NPs) to enter cells and subcellular compartments. A better understanding of these cell entry processes will not only aid in nanomaterial applications but also broaden our knowledge of cell biology. Among the endocytic routes, macropinocytosis has unique characteristics for engulfing NPs and other large cargo, yet its molecular machinery and involvement in NP uptake are far less characterized relative to other pathways. In this review, we summarize the current knowledge on the macropinocytic machinery, and its involvement in NP internalization. Particularly, we differentiate ligand (specifically peptide)-functionalized and unfunctionalized NPs (bystander NPs). While most of previous research focused on ligand-functionalized NPs, we showcase here a synergistic effect between these two NP types during their cell entry through receptor-mediated macropinocytosis. The regulation of NP uptake efficiency by extracellular amino acids is also highlighted in the context of interconnections between macropinocytosis and metabolic signaling. These discussions may fuel future research interests in improving NP internalization through this pathway, and open a new avenue to study the interplay among endocytosis, metabolism and nanomedicine.

Transportan Peptide Stimulates the Nanomaterial Internalization into Mammalian Cells in the Bystander Manner through Macropinocytosis.

Covalent coupling with cell-penetrating peptides (CPPs) has been a common strategy to facilitate the cell entry of nanomaterial and other macromolecules. Though efficient, this strategy requires chemical modifications on nanomaterials, which is not always desired for their applications. Recent studies on a few cationic CPPs have revealed that they can stimulate the cellular uptake of nanoparticles (NPs) simply via co-administration (bystander manner), which bypasses the requirement of chemical modification. In this study, we investigated the other classes of CPPs and discovered that transportan (TP) peptide, an amphiphilic CPP, also exhibited such bystander activities. When simply co-administered, TP peptide enabled the cells to engulf a variety of NPs, as well as common solute tracers, while these payloads had little or no ability to enter the cells by themselves. This result was validated in vitro and ex vivo, and TP peptide showed no physical interaction with co-administered NPs (bystander cargo). We further explored the cell entry mechanism for TP peptide and its bystander cargo, and showed that it was mediated by a receptor-dependent macropinocytosis process. Together, our findings improve the understanding of TP-assisted cell entry, and open up a new avenue to apply this peptide for nanomaterial delivery.

Membrane-curvature-mediated co-endocytosis of bystander and functional nanoparticles.

Efficient cellular uptake of nanoparticles (NPs) is necessary for the development of nanomedicine in biomedical applications. Recently, the coadministration of functionalized NPs (FNPs) was shown to stimulate the cellular uptake of nonfunctionalized NPs (termed bystander NPs, BNPs), which presents a new strategy to achieve synergistic delivery. However, a mechanistic understanding of the underlying mechanism is still lacking. In this work, the bystander uptake effect was investigated at the cell membrane level by combining the coarse-grained molecular dynamics, potential of mean force calculation and theoretical energy analysis methods. The membrane internalization efficiency of BNPs was enhanced by co-administered FNPs, and such activity depends on the affinity of both NPs to the membrane and the resultant membrane deformation. The membrane-curvature-mediated attraction and aggregation of NPs facilitated the membrane uptake of BNPs. Furthermore, quantitative suggestions were given to modulate the BNP internalization through controlling the FNP properties such as size, concentration and surface-ligand density. Our results provide insight into the molecular mechanism of the bystander uptake effect, and offer a practical guide to regulate the cellular internalization of NPs for targeted and efficient delivery to cells.

Cellular internalization of bystander nanomaterial induced by TAT-nanoparticles and regulated by extracellular cysteine.

Entry into cells is necessary for many nanomaterial applications, and a common solution is to functionalize nanoparticles (NPs) with cell-penetrating ligands. Despite intensive studies on these functionalized NPs, little is known about their effect on cellular activities to engulf other cargo from the nearby environment. Here, we use NPs functionalized with TAT (transactivator of transcription) peptide (T-NPs) as an example to investigate their impact on cellular uptake of bystander cargo. We find that T-NP internalization enables cellular uptake of bystander NPs, but not common fluid markers, through a receptor-dependent macropinocytosis pathway. Moreover, the activity of this bystander uptake is stimulated by cysteine presence in the surrounding solution. The cargo selectivity and cysteine regulation are further demonstrated ex vivo and in vivo. These findings reveal another mechanism for NP entry into cells and open up an avenue of studying the interplay among endocytosis, amino acids, and nanomaterial delivery.

Rapid chelator-free radiolabeling of quantum dots for in vivo imaging.

Most current nanoparticle-based PET tracers are radiolabeled through metal chelators conjugated on the nanoparticle surface. Metal chelation usually requires sophisticated optimization and may impact the physical or chemical properties of nanoparticles, which leads to the changes in their distribution and pharmacokinetics in vivo. A chelator-free radiolabeling approach is thus highly desirable. Here, we report that zinc sulfide (ZnS) quantum dots (QDs) can be rapidly radiolabeled with 68Ga or 64Cu through cation exchange without chelators. The radiolabeling was accomplished in times as short as 5 min at 37 °C in aqueous solution, yielding a high labeling efficiency and radiochemical purity for both isotopes. Surface functionalization with targeting peptides was also readily achieved to enable or enhance the cellular uptake of QDs. In vivo PET imaging showed that 64Cu-labeled QDs had a much higher tumor uptake (7.3% ID g-1) than 64Cu-DOTA in a murine cancer model. Overall, this study presents a QD-based platform to achieve convenient and chelator-free radiolabeling, and improve PET imaging of solid tumors.

Tumor-specific macrophage targeting through recognition of retinoid X receptor beta.

Macrophages play important and diverse roles during cancer progression. However, cancer therapies based on macrophage modulation are lacking in tools that can recognize and deliver therapeutic payloads to macrophages in a tumor-specific manner. As a result, treatments tend to interfere with normal macrophage functions in healthy organs. We previously identified a macrophage-binding peptide, termed CRV. Here, we show that upon systemic administration into tumor-bearing mice, CRV selectively homes to tumors, extravasates, and preferentially binds to macrophages within. CRV exhibits a higher affinity for tumor macrophages than for other cells in tumors or for other macrophage types elsewhere in the body. We further identified and validated retinoid X receptor beta (RXRB) as the CRV receptor. Intriguingly, although it is known as a nuclear receptor, RXRB shows a prominent cell surface localization that is largely restricted to tumor macrophages. Systemic administration of anti-RXRB antibodies also results in tumor-selective binding to macrophages similar to CRV. Lastly, we demonstrate the ability of CRV to improve the delivery of nano-carriers into solid tumors and macrophages within. In summary, we describe here a novel cell surface marker and targeting tools for tumor macrophages that may aid in future development of macrophage-modulatory cancer therapies.

Immunogene therapy with fusogenic nanoparticles modulates macrophage response to Staphylococcus aureus.

The incidence of adverse effects and pathogen resistance encountered with small molecule antibiotics is increasing. As such, there is mounting focus on immunogene therapy to augment the immune system's response to infection and accelerate healing. A major obstacle to in vivo gene delivery is that the primary uptake pathway, cellular endocytosis, results in extracellular excretion and lysosomal degradation of genetic material. Here we show a nanosystem that bypasses endocytosis and achieves potent gene knockdown efficacy. Porous silicon nanoparticles containing an outer sheath of homing peptides and fusogenic liposome selectively target macrophages and directly introduce an oligonucleotide payload into the cytosol. Highly effective knockdown of the proinflammatory macrophage marker IRF5 enhances the clearance capability of macrophages and improves survival in a mouse model of Staphyloccocus aureus pneumonia.

Hyperoside protects against hypoxia/reoxygenation induced injury in cardiomyocytes by suppressing the Bnip3 expression.

AIMS: Role of hyperoside in protecting cardiomyocytes from ischemia/reperfusion induced injury has been proved. However, possible protecting mechanisms remain unclear. To fix the problem, an essential pro-apoptotic protein Bnip3 was studied in our experiments. METHODS AND RESULTS: Neonatal rat cardiomyocytes were used and submitted to hypoxia for 8h followed by reoxygenation for 2h to simulate the ischemia/reperfusion injury. Hypoxia/reoxygenation(H/R) induced damage to cardiomyocytes and the protective effect of hyperoside were examined by means of MTT assay. H/R-induced apoptosis was assessed by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL) and DNA Ladder assay. mRNA expression of Bnip3 was determined by use of quantitative real-time reverse transcription polymerase chain reaction assay. Protein levels of Bnip3, Bax, Bcl-2 and cleaved caspase-3 were examined using western-blot assay. Our results showed that H/R caused great damage to cardiomyocytes, upregulated the protein expressions of Bnip3, Bax, cleaved caspase3, and decreased the expression of the anti-apoptotic protein of Bcl-2. Whereas, compared with the H/R group, a decrease in activities of Bnip3, Bax, cleaved caspase3, and a promoting expression of Bcl-2 were detected in the H/R goup pretreated with hyperoside. CONCLUSION: It was concluded in our study that H/R-induced apoptotic effect in cardiomyocytes could be attenuated by hyperoside, and the protective role of hyperoside, if not completely, could be partly through the suppression of the pro-apoptotic gene Bnip3.

Decision-making Impairments in Primary Angle-closure Glaucoma Patients.

BACKGROUND: Primary angle-closure glaucoma (PACG) is a common eye disease and a common cause of blindness. Inappropriate medical decisions severely affect the prognosis. This study investigated decision-making under risk in PACG patients. METHODS: Thirty patients with first acute attack of PACG before surgery and thirty healthy controls were included in the study. Decision-making under risk was evaluated with the game of dice task (GDT). The results of Eysenck Personality Questionnaire (EPQ) and GDT between PACG patients and healthy controls were compared. RESULTS: Risky decisions in PACG patients were more than those in healthy controls as measured by mean score of GDT (12.47 ± 5.72 vs. 4.33 ± 3.30, P< 0.001). Higher neuroticism score in EPQ was found in PACG patients compared to healthy controls (14.97 ± 3.93 vs. 9.90 ± 4.49, P< 0.001). Neuroticism scores in EPQ were associated with decision-making performance (r = 0.417, P = 0.001). CONCLUSIONS: Neuroticism positively correlated with risky decisions. Decision-making might be influenced by neuroticism. Future studies will show whether therapy compliance will be improved by emotional management and psychological intervention in PACG patients.