Additive-free Absorbable Keratin Sponge With Procoagulant Activity for Noncompressible Hemostasis.

The development of a natural, additive-free, absorbable sponge with procoagulant activity for noncompressible hemostasis remains a challenging task. In this study, we extracted high molecular weight keratin (HK) from human hair and transformed it into a hemostatic sponge with a well-interconnected pore structure using a foaming technique, freeze-drying, and oxidation cross-linking. By controlling the cross-linking degree, the resulting sponge demonstrated excellent liquid absorption ability, shape recovery characteristics, and robust mechanical properties. The HK10 sponge exhibited rapid liquid absorption, expanding up to 600% within 5 s. Moreover, the HK sponge showed superior platelet activation and blood cell adhesion capabilities. In SD rat liver defect models, the sponges demonstrated excellent hemostatic performance by sealing the wound and expediting coagulation, reducing the hemostatic time from 825 to 297 s. Furthermore, HK sponges have excellent biosafety, positioning them as a promising absorbable sponge with the potential for the treatment of noncompressible hemostasis.

Oligohistidine and targeting peptide functionalized TAT-NLS for enhancing cellular uptake and promoting angiogenesis in vivo.

BACKGROUND: Gene therapy has been developed and used in medical treatment for many years, especially for the enhancement of endothelialization and angiogenesis. But slow endosomal escape rate is still one of the major barriers to successful gene delivery. In order to evaluate whether introducing oligohistidine (Hn) sequence into gene carriers can promote endosomal escape and gene transfection or not, we designed and synthesized Arg-Glu-Asp-Val (REDV) peptide functionalized TAT-NLS-Hn (TAT: typical cell-penetrating peptide, NLS: nuclear localization signals, Hn: oligohistidine sequence, n: 4, 8 and 12) peptides with different Hn sequence lengths. pEGFP-ZNF580 (pZNF580) was condensed by these peptides to form gene complexes, which were used to transfect human umbilical vein endothelial cells (HUVECs). RESULTS: MTT assay showed that the gene complexes exhibited low cytotoxicity for HUVECs. The results of cellular uptake and co-localization ratio demonstrated that the gene complexes prepared from TAT-NLS-Hn with long Hn sequence (n = 12) benefited for high internalization efficiency of pZNF580. In addition, the results of western blot analysis and PCR assay of REDV-TAT-NLS-H12/pZNF580 complexes showed significantly enhanced gene expression at protein and mRNA level. Wound healing assay and transwell migration assay also confirmed the improved proliferation and migration ability of the transfected HUVECs by these complexes. Furthermore, the in vitro and in vivo angiogenesis assay illustrated that these complexes could promote the tube formation ability of HUVECs. CONCLUSION: The above results indicated that the delivery efficiency of pZNF580 and its expression could be enhanced by introducing Hn sequence into gene carriers. The Hn sequence in REDV-TAT-NLS-Hn is beneficial for high gene transfection. These REDV and Hn functionalized TAT-NLS peptides are promising gene carriers in gene therapy.

Core/Shell Gene Carriers with Different Lengths of PLGA Chains to Transfect Endothelial Cells.

In order to improve the transfection efficiency and reduce the cytotoxicity of gene carriers, many strategies have been used to develop novel gene carriers. In this study, five complex micelles (MSP(2 k), MSP(4 k), MSP(6 k), MSP(8 k), and MSP(10 k)) were prepared from methoxy-poly(ethylene glycol)-b-poly(d,l-lactide-co-glycolide) (mPEG-b-PLGA) and sorbitol-poly(d,l-lactide-co-glycolide)-graft-PEI (sorbitol-PLGA-g-PEI, where the designed molecular weights of PLGA chains were 2 kDa, 4 kDa, 6 kDa, 8 kDa, and 10 kDa, respectively) copolymers by a self-assembly method, and the mass ratio of mPEG-b-PLGA to sorbitol-PLGA-g-PEI was 1/3. These complex micelles and their gene complexes had appropriate sizes and zeta potentials, and pEGFP-ZNF580 (pDNA) could be efficiently internalized into EA.hy926 cells by their gene complexes (MSP(2 k)/pDNA, MSP(4 k)/pDNA, MSP(6 k)/pDNA, MSP(8 k)/pDNA, and MSP(10 k)/pDNA). The MTT assay results demonstrated that the gene complexes had low cytotoxicity in vitro. When the hydrophobic PLGA chain increased above 6 kDa, the gene complexes showed higher performance than that prepared from short hydrophobic chains. Moreover, the relative ZNF580 protein expression levels in MSP(6 k)/pDNA, MSP(8 k)/pDNA, and MSP(10 k)/pDNA) groups were 79.6%, 71.2%, and 73%, respectively. These gene complexes could promote the transfection of endothelial cells, while providing important information and insight for the design of new and effective gene carriers to promote the proliferation and migration of endothelial cells.

Multitargeting Gene Delivery Systems for Enhancing the Transfection of Endothelial Cells.

Gene therapy demonstrates promising prospects on cardiovascular diseases. However, nonviral gene delivery system has relatively low transfection efficiency, especially for endothelial cells (ECs). Herein, typical cell-penetrating peptide (TAT), nuclear localization signals (NLSs), and REDV functional peptide have been used to prepare multitargeting complexes. These complexes exhibit higher transfection efficiency owing to the targeting sequences of REDV and NLSs as well as the cell-penetrating function of TAT. The multifunction of the complexes provides high cell uptake, endo/lysosomal escape, and nucleus accumulation of the encapsulated DNA. Thus these multitargeting complexes can provide a potential platform for gene delivery, especially for EC transfection.

A composite hydrogel loaded with the processed pyritum promotes bone repair via stimulate the osteogenic differentiation of BMSCs.

Tissue engineering shows promise in repairing extensive bone defects. The promotion of proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by biological scaffolds has a significant impact on bone regeneration outcomes. In this study we used an injectable hydrogel, known as aminated mesoporous silica gel composite hydrogel (MSNs-NH2@GelMA), loaded with a natural drug, processed pyritum (PP), to promote healing of bone defects. The mechanical properties of the composite hydrogel were significantly superior to those of the blank hydrogel. In vitro experiments revealed that the composite hydrogel stimulated the osteogenic differentiation of BMSCs, and significantly increased the expression of type I collagen (Col 1), runt-related transcription factor 2 (Runx 2), alkaline phosphatase (ALP), osteocalcin (OCN). In vivo experiments showed that the composite hydrogel promoted the generation of new bones. These findings provide evidence that the composite hydrogel pyritum-loaded holds promise as a biomaterial for bone repair.

Expression of concern: Poly(lactide-co-glycolide) grafted hyaluronic acid-based electrospun fibrous hemostatic fragments as a sustainable anti-infection and immunoregulation material.

Expression of concern for 'Poly(lactide-co-glycolide) grafted hyaluronic acid-based electrospun fibrous hemostatic fragments as a sustainable anti-infection and immunoregulation material' by Wen Liu et al., J. Mater. Chem. B, 2019, 7, 4997-5010, https://doi.org/10.1039/C9TB00659A.

Gelatin Methacryloyl-Based Sponge with Designed Conical Microchannels for Rapidly Controlling Hemorrhage and Theoretical Verification.

It remains a challenge to develop effective hemostatic products in battlefield rescue for noncompressible massive hemorrhage. Some previous research had concentrated on the modification of different materials to improve the hemostasis ability of sponges. Herein, to investigate the relationship between the taper of microchannels and hemostatic performance of porous sponges, gelatin methacryloyl-based sponges with designed conical microchannels and a disordered porous structure were prepared using the 3D printing method and freeze-drying technology. Experiments and theoretical model analysis demonstrated that the taper and distribution of microchannels in the sponge affected the water and blood absorption properties, as well as the expansion ability. In treatment of SD rat liver defect and SD rat liver perforation wound, GS-1 sponge with the taper (1/15) microchannels exhibited an excellent hemostatic effect with blood loss of 0.866 ± 0.093 g and a hemostasis time of 280 ± 10 s. Results showed that the hemostatic capacities of GelMA sponges were increased with the bottom diameter (taper) of conical microchannels. This is a potential strategy to develop designed taper sponges with designed taper microchannels for rapidly controlling hemorrhage.

Photo-controllable burst generation of peroxynitrite based on synergistic interactions of polymeric nitric oxide donors and IR780 for enhancing broad-spectrum antibacterial therapy.

The newly attractive peroxynitrite (ONOO-) therapy can prominently enhance antibacterial therapeutic efficacy. However, it is a great challenge but urgently needed to generate ONOO- with adjustable release rate and dosage in order to satisfy personalized treatments for different disease types and severities. Herein, PSNO@IR780 nanoparticles are fabricated via co-assembly of an amphiphilic PEG-b-PAASNO block copolymer grafted with abundant nitric oxide (NO) donor units and IR780 as a photothermal and photodynamic agent. Photo-controllable burst generation of ONOO- from PSNO@IR780 nanoparticles could be realized based on synergistic reactions of rapid NO release induced by increased local temperature and efficiently produced superoxide anion radical (O2•-) from IR780. The maximum ONOO- release dosage is up to 6.73 ± 0.07 µM and release rate is up to 98.1 ± 1.38 nM/s. Furthermore, the ONOO- release behavior can be precisely manipulated by varying sample concentrations, irradiated durations, output power densities, and laser switches, respectively. Ultra-efficiently generated ONOO- from biocompatible PSNO@IR780 nanoparticles significantly elevated broad spectrum antibacterial efficiency through damaging bacterial membranes. Thus, PSNO@IR780 nanoparticles may present a new insight into preparation of burst and controllable generating ONOO- materials, and provide new opportunities for antibacterial therapy. STATEMENT OF SIGNIFICANCE: 1. Polymeric NO donor (PEG-b-PAASNO) grafted with abundant NO donor units was synthesized. 2. PSNO@IR780 nanoparticles were prepared by co-assembly of IR780 and amphiphilic PEG-b-PAASNOpolymer. 3. The maximum ONOO- release dosage from PSNO@IR780 nanoparticles was 6.73 ± 0.08 µM. 4. The fastest ONOO- release rate from PSNO@IR780 nanoparticles was 98.1 ± 1.4 nM/s. 5. Ultra-efficiently generated ONOO- significantly elevated antibacterial efficiency via damaging bac-terial membranes.

Polydopamine functionalized polyurethane shape memory sponge with controllable expansion performance triggered by near-infrared light for incompressible hemorrhage control.

Uncontrolled expansion of shape memory sponges face a significant challenge in the treatment of lethal incompressible hemorrhage, which can lead to blood overflow or damage to the surrounding tissue. Herein, we developed a polydopamine functionalized polyurethane shape memory sponge (PDA-TPI-PU) with a controllable degree of expansion by near-infrared (NIR) light-triggered stimulation for the treatment of incompressible hemorrhage. The sponge has excellent liquid absorption performance and robust mechanical strength as well as good photothermal conversion ability. Under NIR light of 0.32 W/cm2, the maximum recovery rate of the fixed-shape compression sponge was 91% within 25 s in air and 80% within 25 s in blood. In the SD rat liver penetrating injury model, compared with commercial medical gelatin sponge and PVA sponge, the PDA-TPI-PU sponge could effectively control the bleeding under the NIR light irradiation and did not cause excessive compression of the wound. The sponge with these characteristics shows potential application prospects as a hemostatic material.

Wool keratin/zeolitic imidazolate framework-8 composite shape memory sponge with synergistic hemostatic performance for rapid hemorrhage control.

Uncontrollable hemorrhage and subsequent wound infection pose severe threats to life, especially in the case of deep, non-compressible, massive bleeding. Here, a wool keratin/zeolitic imidazolate framework-8 (WK/ZIF-8) composite shape memory sponge is prepared by incorporating ZIF-8 nanoparticles into wool keratin. The combination of keratin and ZIF-8 particles not only reduces the effect of ZIF-8 particles on cell viability but also bolsters the mechanical properties of the keratin sponge and endows it with antibacterial efficacy. Due to the synergistic effect of the excellent hemostatic performance of keratin and Zn2+ release from ZIF-8 nanoparticles, the porous structure suitable for blood cell adhesion and the shape recovery ability of sponges, the WK/ZIF-8 composite sponge exhibits superior hemostatic performance to commercial medical sponges in SD rat and rabbit hemorrhage models. In addition, in vitro and in vivo antibacterial experiments demonstrate the anti-infection activity of the composite sponge. Overall, the WK/ZIF-8 composite sponge provides a promising approach to rapidly control bleeding and promote wound healing.

A graphene oxide-loaded processed pyritum composite hydrogel for accelerated bone regeneration via mediation of M2 macrophage polarization.

A coordinated interaction between osteogenesis and the osteoimmune microenvironment plays a vital role in regulating bone healing. However, disturbances in the pro- and anti-inflammatory balance hinder the therapeutic advantages of biomaterials. In this study, a novel composite hydrogel was successfully fabricated using graphene oxide (GO)-loaded processed pyritum (PP) in combination with poly(ethylene glycol) diacrylate (PEGDA) and carboxymethyl chitosan (CMC). Subsequently, the immunomodulatory effects and bone regenerative potential of PP/GO@PEGDA/CMC were investigated. The results demonstrated that the PP/GO@PEGDA/CMC hydrogel possessed excellent mechanical properties, swelling capacity, and stability. Moreover, PP/GO@PEGDA/CMC prominently promoted M2 polarization and increased the levels of anti-inflammatory factors (interleukin (IL)-4, IL-10, and transforming growth factor-ß). These beneficial effects facilitated the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells in vitro. Additionally, the in vivo results further verified that the implantation of PP/GO@PEGDA/CMC markedly reduced local inflammation while enhancing bone regeneration at 8 weeks post-implantation. Therefore, the results of this study provide potential therapeutic strategies for bone tissue repair and regeneration by modulating the immune microenvironment.

Expandable, biodegradable, bioactive quaternized gelatin sponges for rapidly controlling incompressible hemorrhage and promoting wound healing.

Designing expandable sponges with biodegradability and effective antibacterial properties are the urgent challenge for incompressible hemorrhage and wound healing. In the present investigation, based on quaternized gelatin (QG) and oxidized dextran (OD), a series of expandable sponges (ODQG) with high-water absorption capacity and robust mechanical properties were prepared. ODQG had good biodegradability in vitro and in vivo, and had inherent antibacterial activity (90% for E. coli and 99.74% for S. aureus). Due to the synergy effect of electrostatic interaction and blood concentration, ODQG could effectively attract and activate red blood cells/platelets and accelerate the coagulation process. Therefore, ODQG showed better hemostatic performance than Kuaikang® gelatin sponges and gauzes in incompressible hemorrhage model. Furthermore, ODQG could regulate inflammatory factor (TNF-α) and cytokines (TGF-ß, VEGF), and greatly promote wound healing process. The biodegradable sponges with excellent antibacterial properties might have potential application prospect for incompressible hemostasis and wound healing in the future.

Polyvinyl alcohol/sodium alginate composite sponge with 3D ordered/disordered porous structure for rapidly controlling noncompressible hemorrhage.

It is still a challenge to develop a sponge that can efficiently control noncompressible bleeding to meet the emergency treatment and clinical demand. Herein, we combined the 3D printing sacrificial template method and freeze-drying technology to prepare polyvinyl alcohol/sodium alginate (PVA/SA) composite sponges with ordered microchannels and disordered porous structure. Compared with conventional sponges, the prepared sponge showed ultra-rapid water/blood absorption capacity and satisfactory mechanical properties. Furthermore, when the sponge was stuffed into a noncompressible wound and contacted with blood, it could accurately guide and quickly absorb a large amount of blood through the microchannels. Moreover, the platelets, red blood cells and coagulation factors would be enriched in the microchannels and microporous structure. In the SD rat liver noncompressible hemorrhage and femoral artery puncture injury model, PVA-SA composite sponge with 3D ordered/disordered porous structure showed enhanced hemostatic performance compared with commercial MPVA sponges. Depend on the special ordered/disordered porous structure, PVA-SA composite sponge could accelerate the blood convergence and promote coagulation. This design of special porous structure opened up a new avenue to develop hemostatic sponges for rapidly controlling noncompressible hemorrhage.

Lipase-triggered drug release from BCL2 inhibitor ABT-199-loaded nanoparticles to elevate anti-leukemic activity through enhanced drug targeting on the mitochondrial membrane.

Selective BCL2 inhibitor ABT-199 has been approved to treat hematological malignancies including acute myeloid leukemia (AML). However, acquired drug resistance and severe side effects occur after extended treatment limiting the clinical usage of ABT-199. Here, we successfully encapsulated pure ABT-199 in amphiphilic mPEG-b-PTMC169 block copolymer, forming mPEG-b-PTMC169@ABT-199 nanoparticles (abbreviated as PEG-ABT-199), which presented better aqueous dispersion and higher efficiency of loading and encapsulation than pure ABT-199. We then compared the anti-leukemic ability of pure ABT-199 and PEG-ABT-199 in vitro and in vivo. PEG-ABT-199 had a lower IC50 value compared with pure ABT-199 in MV4-11 and MOLM-13 cell lines. In addition, PEG-ABT-199 significantly induced apoptosis and decreased colony number than pure ABT-199. Most importantly, PEG-ABT-199 markedly reduced leukemic burden, inhibited the infiltration of leukemic blasts in the spleen, and extended the overall survival (OS) in MLL-AF9-transduced murine AML compared with free ABT-199. Meanwhile, the blank PEG169 NP was non-toxic to normal hematopoiesis in vitro and in vivo, suggesting that PEG169 NP is a safe carrier. Mechanistically, PEG-ABT-199 enhanced mitochondria-targeted delivery of ABT-199 to trigger the collapse of mitochondrial membrane potential (MMP), the release of cytochrome c (cyt-c), and mitochondria-based apoptosis. In conclusion, our results suggest that PEG-ABT-199 has more vital anti-leukemic ability than pure ABT-199. PEG-ABT-199 has potential application in clinical trials to alleviate side effects and improve anti-leukemia ability. STATEMENT OF SIGNIFICANCE: ATB-199, an orally selective inhibitor for BCL2 protein, presents marked activity in relapsed or refractory AML, T-ALL, and CLL patients. However, ABT-199 resistance severely limits the further clinical usage because of off-target effects, non-specific toxicities, and low delivery of drugs. To reduce the side-effects and improve the solubility and bioavailability, ABT-199 was encapsulated into the amphiphilic mPEG-b-PTMC block copolymer by co-assembly method to obtain mPEG-b-PTMC@ABT-199 nanoparticles (PEG-ABT-199). PEG-ABT-199 has several advantages compared with pure ABT-199. 1.PEG-ABT-199 presents better aqueous dispersion and higher efficiencies of loading and encapsulation than pure ABT-199. 2. PEG-ABT-199 substantially enhances the anti-leukemic ability in vitro and in vivo compared with pure ABT-199. 3. PEG-ABT-199 has little effects on normal cells. 4. PEG-ABT-199 can reduce treatment cost.

Fabricating poly(vinyl alcohol)/gelatin composite sponges with high absorbency and water-triggered expansion for noncompressible hemorrhage and wound healing.

It is challenging for traditional hemostatic sponges to control massive and noncompressible hemorrhages in the military field and accidental trauma. In this work, a series of highly fluid-absorbent composite sponges with rapid expansion ability based on norbornene anhydride-modified poly(vinyl alcohol) and gelatin (PVA@Gel-Sps) were developed by a foaming technique, chemical and physical crosslinking reactions and lyophilization. The prepared PVA@Gel-Sp2 exhibited a 3500% maximum water absorption ratio with a fast water absorption speed, which was suitable for blood component concentration. Owing to its interconnected macroporous structure, robust mechanical strength and high resilience, the compressed sponge could rapidly re-expand to more than 10 times its volume in response to water and blood. Moreover, due to the synergistic effect of the PVA-based sponge and gelatin, PVA@Gel-Sp2 could obviously shorten the hemostasis time and reduce blood loss in SD rat liver defect noncompressible hemorrhage models, and exhibited better wound healing effects in a full-thickness skin defect model than commercial sponges. These results suggest that PVA@Gel-Sp2 is a potential candidate for controlling noncompressible hemorrhage and promoting wound healing.

Poly(trimethylene carbonate) flexible intestinal anastomosis scaffolds to reduce the probability of intestinal fistula and obstruction.

Biodegradable anastomat play an important role in the reconstruction process of the digestive tract. However, the biocompatibility and organizational compliance of anastomotic tubes still need to be improved. Electrospun tissue engineering scaffolds have excellent biomimetic extracellular matrix properties, biocompatibility and biodegradability. In the present study, electrospun poly(trimethylene carbonate) (PTMC) intestinal anastomosis scaffolds loaded with triclosan (TCS) were reported to reduce the probability of intestinal fistula and obstruction. When the viscosity average molecular weight of PTMC was 157 × 103, the elastic modulus and tensile strength of the anastomosis scaffolds could reach 20.11 MPa and 16.08 MPa, respectively, which indicated that the anastomosis scaffolds exhibited excellent tensile flexibility. The degradation of PTMC was accelerated with the increase of Mw. After 28 days, the weight and length of the anastomosis scaffolds reduced 40% and 50%, respectively. Furthermore, the application of PTMC anastomosis scaffolds could promote intestinal healing and reduce the probability of intestinal fistula and obstruction.

Thrombin Embedded in eMPs@Thr/Sponge with Enhanced Procoagulant Ability for Uncompressible and Massive Hemorrhage Control.

Uncontrollable hemorrhage, especially uncompressible and massive hemorrhage, is life threatening. To develop a kind of hemostatic material that is biocompatible and has effective hemostatic performance, a starch-polyethylene glycol sponge (St-PEG sponge) incorporated with electrospraying microspheres made from carboxymethyl chitosan and sodium alginate (CMC/SA eMPs) is employed to fabricate the topical hemostatic agent for application. To load thrombin safely and effectively, CMC/SA eMPs encapsulating thrombin compounds (eMPs@Thr) are further incorporated with St-PEG sponge to obtain eMPs@Thr/sponge. The results show that eMPs@Thr/sponge could obviously reduce blood loss and shorten the hemostatis time compared with commercial hemostatic products Kuai Kang. The construction of the eMPs@Thr/sponge could not only maintain the expandable properties of the St-PEG sponge but also strengthen the procoagulant activity. Therefore, this work provides a facile approach for loading thrombin given the fact that thrombin suffers from instability and risk of thrombus. It is predicted that eMPs@Thr/sponge might hold great potential in uncompressible and massive hemorrhage control.

Controlled release of KGF-2 for regulation of wound healing by KGF-2 complexed with "lotus seedpod surface-like" porous microspheres.

Keratinocyte growth factor-2 (KGF-2) can regulate the proliferation and differentiation of keratinocyte, which plays a remarkable role in maintaining normal tissue structure and promoting wound healing. As an effective strategy, KGF-2 solution is widely used in the treatment of wounds in clinical applications. However, KGF-2 in solution cannot achieve sustained release, which results in drug loss and unnecessary waste. Polysaccharide hemostasis microspheres (PHMs) are an ideal drug loading platform due to their special "lotus seedpod surface-like" morphology and structure. Herein, to realize the controllable release of KGF-2, PHMs loaded with KGF-2 (KGF-2@PHMs) were prepared. It was found that the bioavailability of KGF-2 was improved greatly. Most importantly, KGF-2@PHMs can reduce inflammation and accelerate the wound healing process due to the controlled release of KGF-2. KGF-2@PHMs might be a potential alternative strategy for wound healing in future clinical applications.

Hydrodynamic cavitation: A feasible approach to intensify the emulsion cross-linking process for chitosan nanoparticle synthesis.

Chitosan nanoparticles (NPs) exhibit great potential in drug-controlled release systems. A controlled hydrodynamic cavitation (HC) technique was developed to intensify the emulsion crosslinking process for the synthesis of chitosan NPs. Experiments were performed using a circular venturi and under varying operating conditions, i.e., types of oil, addition mode of glutaraldehyde (Glu) solution, inlet pressure (Pin), and rheological properties of chitosan solution. Palm oil was more appropriate for use as the oil phase for the HC-intensified process than the other oil types. The addition mode of water-in-oil (W/O) emulsion containing Glu (with Span 80) was more favorable than the other modes for obtaining a narrow distribution of chitosan NPs. The minimum size of NPs with polydispersity index of 0.342 was 286.5 nm, and the maximum production yield (Py) could reach 47.26%. A positive correlation was found between the size of NPs and the droplet size of W/O emulsion containing chitosan at increasing Pin. Particle size, size distribution, and the formation of NPs were greatly dependent on the rheological properties of the chitosan solution. Fourier transform infrared spectroscopy (FTIR) analysis indicated that the molecular structure of palm oil was unaffected by HC-induced effects. Compared with ultrasonic horn, stirring-based, and conventional drop-by-drop processes, the application of HC to intensify the emulsion crosslinking process allowed the preparation of a finer and a narrower distribution of chitosan NPs in a more energy-efficient manner. The novel route developed in this work is a viable option for chitosan NP synthesis.

The quantitative research of composite immune indicator for crustacean.

Through analyzing the immunity indicators in recent crustacean research, two defects are pointed in comprehensive immunity evaluation, 1) the integrant indicators cannot comprehensively reflect the change of immunity, and 2) the conclusions that obtained from different indicators of immunity level cannot be compared objectively and scientifically. Basing on that, the paper firstly indicated that the immunity system could be regarded as a composite indicator. Secondly, the paper gave the specific definition of the composite immunity indicator (CII), and discussed the methods of calculation, especially provided two calculation methods of the weights, that is, the Analytic Hierarchy Process (AHP) and the Principal Component Analysis (PCA). Finally, examples were given to clarify the specific steps to compute the composite immunity indicator. The computing results gave the quantitative evaluation, which were in concordance with the existing conclusions.