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Surgery is an important therapeutic strategy for intracerebral hemorrhage (ICH) in the clinic and is theoretically beneficial for the outcome of ICH by decreasing hematoma, reducing nervous tissue damage, and removing harmful chemicals. However, the outcome of ICH surgery is always unsatisfactory due to postoperative rebleeding. We hypothesized that the injection of hemostatic agents in situ after aspiration surgery could immediately activate hemostasis once rebleeding occurs. Therefore, keratin hydrogels (K-gels) were easily prepared as a hemostatic material via a rehydration method and had a porous structure. Collagenase was injected into the basal lamina to mimic ICH rebleeding, and the K-gels were then injected into the same injured site after 2 h for hemostatic therapy. The hematoma volume was significantly reduced by K-gel treatment, indicating that in situ infusion of the K-gels inhibited hematoma enlargement when rebleeding occurred. Moreover, brain damage, including cell apoptosis, neuroinflammatory reactions, and neurological deficits, was also relieved after K-gel treatment. These results suggested that in situ injection of the K-gels into the hematoma area after ICH surgery improves the therapeutic outcome by stopping postoperative rebleeding. K-gels have great potential for clinical hemostatic application because of their excellent hemostatic properties and biocompatibility.

Keratins are considered ideal candidates as hemostatic agents, but the development lags far behind their potentials due to the poorly understood hemostatic mechanism and structure-function relations, owing to the composition complexity in protein extracts. Here, it is shown that by using a recombinant synthesis approach, individual types of keratins can be expressed and used for mechanism investigation and further high-performance keratin hemostatic agent design. In the comparative evaluation of full-length, rod-domain, and helical segment keratins, the 𝜶-helical contents in the sequences are identified to be directly proportional to keratins' hemostatic activities, and Tyr, Phe, and Gln residues at the N-termini of 𝜶-helices in keratins are crucial in fibrinopeptide release and fibrin polymerization. A feasible route to significantly enhance the hemostatic efficiency of helical keratins by mutating Cys to Ser in the sequences for enhanced water wettability through soluble expression is then further presented. These results provide a rational strategy to design high-efficiency keratin hemostatic agents with superior performance over clinically used gelatin sponge in multiple animal models.

H uman s erum a lbumin (HSA) is a highly water-soluble protein with 67% alpha-helix content and three distinct domains (I, II, and III). HSA offers a great promise in drug delivery with enhanced permeability and retention effect. But it is hindered by protein denaturation during drug entrapment or conjugation that result in distinct cellular transport pathways and reduction of biological activities. Here we report using a protein design approach named r everse- QTY (rQTY) code to convert specific hydrophilic alpha-helices to hydrophobic to alpha-helices. The designed HSA undergo self-assembly of well-ordered nanoparticles with highly biological actives. The hydrophilic amino acids, asparagine (N), glutamine (Q), threonine (T), and tyrosine (Y) in the helical B-subdomains of HSA were systematically replaced by hydrophobic leucine (L), valine (V), and phenylalanine (F). HSA rQTY nanoparticles exhibited efficient cellular internalization through the cell membrane albumin binding protein GP60, or SPARC ( s ecreted p rotein, a cidic and r ich in c ysteine)-mediated pathways. The designed HSA rQTY variants displayed superior biological activities including: i) encapsulation of drug doxorubicin, ii) receptor-mediated cellular transport, iii) tumor cell targeting, and iv) antitumor efficiency compare to denatured HSA nanoparticles. HSA rQTY nanoparticles provided superior tumor targeting and antitumor therapeutic effects compared to the albumin nanoparticles fabricated by antisolvent precipitation method. We believe that the rQTY code is a robust platform for specific hydrophobic modification of functional hydrophilic proteins with clear-defined binding interfaces.

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