Biomedical subjects

Victor C Yang

Publications and source records attributed to Victor C Yang.

17 recordsLinked to original sources

PubMed2006

Chemically surface modified gel (CSMG): an excellent enzyme-immobilization matrix for industrial processes.

Invertase from S. cerevisiae has been immobilized on porous silica matrix, formed using sol-gel chemistry, with surface area of approximately 650 m(2)/g. The co-condensation of silica sol with 3-aminopropyl(triethoxy)silane produced an amino-chemically surface modified silica gel (N-CSMG) with a very high ligand loading of 3.6 mmol/g SiO(2); significantly higher than commercially available matrices. Surface amine groups were activated with glutaraldehyde to produce GA-N-CSMG, and invertase covalently attached by the aldehyde. Invertase was used as a model enzyme to measure the immobilizing character of the GA-N-CSMG material. Using an optimized immobilization protocol, a very high loading of 723 mg invertase per gram GA-N-CSMG is obtained; 3-200-fold higher than values published in literature. The reproducible, immobilized activity of 246,000 U/g GA-N-CSMG is also greater than any other in literature. Immobilized invertase showed almost 99% retention of free enzyme activity and no loss in catalytic efficiency. The apparent kinetic parameters K(M) and V(M) were determined using the Michealis-Menten kinetic model. K(M) of the free invertase was 1.5 times greater than that of the immobilized invertase--indicating a higher substrate affinity of the immobilized invertase. These findings show considerable promise for this material as an immobilization matrix in industrial processes.

Chemical Industry↗

PubMed2005

A GPIIb/IIIa bioreactor for specific treatment of immune thrombocytopenic purpura, an autoimmune disease. Preparation, in vitro characterization, and preliminary proof-of-concept animal studies.

Immune thrombocytopenic purpura (ITP) is an autoimmune disease that affects thousands of Americans each year. The resulting thrombocytopenia, which develops from destruction of platelets (PLT) by anti-PLT autoantibodies (APAb), is often associated with hemorrhagic complications. Existing therapies are not effective and are associated with significant morbidity. Recently, a new treatment modality using plasmapheresis with a Protein-A column has shown some clinical promise. Yet, although this method would remove the pathogenic APAb, it would also deplete protective antibodies, thereby weakening the body's self-defense system. Because about 80% of patients with ITP develop APAb against the GPIIb/IIIa antigens on PLT, a novel approach of attaching a GPIIb/IIIa-linked bioreactor with an extracorporeal circuit is suggested herein to achieve highly effective/specific APAb removal and overcome shortcomings of plasmapheresis in treating ITP. A hollow fiber-based bioreactor device was fabricated, and GPIIb/IIIa antigens were immobilized onto the inner lumens of the hollow fibers by using the epichlorohydrin activation method. An optimized bioreactor containing a loading of 1.63 mg GPIIb/IIIa/g fibers and adsorption capacity of 1.9 mg 7E3/g fibers was developed. Preliminary proof-of-concept investigation using a 7E3-induced thrombocytopenic rat model (which mimicked clinical ITP) was carried out. A complete (100%) return of PLT counts to their initial levels was observed in rats within 6 h after the GPIIb/IIIa bioreactor treatment. In addition, a rapid restoration of WBC counts in the treated rats was also found. These preliminary findings shed light of promise of using the GPIIb/IIIa bioreactor approach in achieving highly improved ITP therapy.

Animals↗

PubMed2005

Synthesis of doxorubicin-peptide conjugate with multidrug resistant tumor cell killing activity.

Cell penetrating peptide TAT was introduced into doxorubicin structure. Synthesized doxorubicin-TAT conjugate showed different intracellular distribution pattern and cell killing activity from those of free doxorubicin. Unlike free doxorubicin, doxorubicin-TAT conjugate was highly permeable to drug-resistant cells and was able to kill drug-resistant tumor cells efficiently.

Antineoplastic Agents↗

PubMed2005

Construction and characterization of a t-PA mutant for use in ATTEMPTS: a drug delivery system for achieving targeted thrombolysis.

To resolve the bleeding risk associated with thrombolytic therapy, we have designed an approach, termed ATTEMPTS (Antibody Targeted Triggered Electrically Modified Prodrug Type Strategy), to deliver t-PA to the clot site in an inactive form and then trigger its conversion to the active form, so that it would selectively activate the clot bound plasminogen while alleviating the bleeding risk. This delivery system was composed of a large protein complex, consisting of two components: (i) a heparin-modified, negatively charged fibrin-targeting antibody; and (ii) a cationic peptide-modified, positively charged t-PA. Both in vitro and in vivo studies have confirmed the feasibility of this targeted drug delivery approach. A site-specific thrombolysis was observed in animals, without concomitant depletion of the coagulation factors -- the phenomenon in conventional thrombolytic therapy that contributes to the bleeding risk. Despite promise, the chemical conjugation method employed previously in the preparation of the cationic peptide-modified t-PA also revealed several major shortcomings. The primary drawback was that the number of the cationic peptides and the location at which these peptides were attached to a t-PA molecule could not be regulated by using the chemical conjugation method. As a consequence, the resultant modified t-PA possessed a wide range of heparin-binding strength, rendering the inhibition of t-PA activity by heparin binding ineffective. In this paper, we present a new strategy in producing the desired modified t-PA, utilizing the genetic engineering approach. A computer simulation-guided rational design strategy was adopted to identify the most desirable site in t-PA (i.e. the 37-loop) for incorporation of the heparin-binding peptide sequence. By altering the amino acid composition via mutation at three locations, i.e. Ser(300) to Cys, Gly(302) to Arg, and Glu(303) to Arg, a highly cationic nanomer sequence consisting of (297)KHRRCPRRR(304) and possessing a well-demonstrated heparin-binding domain was established within the 37-loop. To ensure the binding of heparin to this specifically modified domain, a cysteine residue (i.e. Cys(300)) was created to allow for site-specific conjugation of an additional heparin-binding peptide (i.e. the LMWP peptide previously developed in our laboratory) to this domain via the chemical conjugation method. In vitro fibrinolysis assays showed that both the t-PA mutant and the LMWP-attached t-PA mutant exhibited a fibrinolytic potency similar to that of the wild type t-PA. Inhibition studies using small chromogenic substrates demonstrated that the activity of mutant tPA-LMWP could be significantly inhibited by heparin binding. In conclusion, using computer simulation and molecular biology approaches, a mutated t-PA that meets the needs of the ATTEMPTS system, in providing a safe thrombolytic therapy, could be readily prepared.

Animals↗

PubMed2005

Strategies for improving the functionality of an affinity bioreactor.

Heparin employed in extracorporeal blood circulation (ECBC) procedures (e.g. open heart operations) often leads to a high incidence of bleeding complications. Protamine employed in heparin neutralization, on the other hand, can cause severe adverse reactions. We previously developed an approach that could prevent both heparin- and protamine-induced toxic side effects concomitantly. This approach consisted of placing a hollow fiber-based bioreactor device containing immobilized protamine (termed a "protamine bioreactor") at the distal end of the ECBC procedure. This protamine bioreactor would remove heparin after heparin served its anticoagulant purpose in the ECBC device, thereby eliminating heparin-induced bleeding risks. In addition, this protamine bioreactor would prevent protamine from entering the patients, thereby aborting any protamine-induced toxic effects. Both in vitro and in vivo studies have successfully demonstrated the feasibility of this approach. Despite promises, early findings also revealed two shortcomings that must be overcome for the protamine bioreactor to be applied clinically. The first drawback was that the cyanate ester linkages, involved in conjugating protamine to the bioreactor device, were unstable and prone to hydrolysis, resulting in the leakage of a significant amount of protamine into circulation during application of the protamine bioreactor. The second deficiency was that the capacity of the protamine bioreactor in heparin removal was rather low, owing to the limited surface area of the hollow fibers for protamine immobilization and subsequently heparin adsorption. In this paper, we present novel strategies to overcome these two limitations. A new conjugation method based on the use of 4-(oxyacetyl)phenoxyacetic acid (OAPA) as the activating reagent was employed to yield stable linkages, via the abundant arginine residues of protamine, onto the hollow fibers. Results showed that while the amount of protamine immobilized on each gram of fibers was relatively comparable between the OAPA and the previous CNBr activation methods (7.45 mg/g versus 7.69 mg/g fibers), there was virtually no detectable leaching of immobilized protamine from the bioreactor by the OAPA method, comparing to 35% leaching of protamine by the previous CNBr method following 72 h of storage of the bioreactor in PBS buffer at 37 degrees C. To improve the capacity and functionality of the protamine bioreactor, two novel approaches were adopted. Long chain and high molecular weight poly-lysine was linked to the hollow fibers, prior to protamine coupling, to create multiple layers of immobilized protamine for subsequent heparin adsorption. In addition, a poly(ethylene glycol) (PEG) chain was inserted between protamine and the hollow fibers to yield a three-dimensional, free dynamic motion for immobilized protamine. Preliminary observations indicated that a four- to five-fold enhancement in heparin adsorption was attained by utilizing each of these new approaches. Aside from their current use, these new strategies can also be employed generically to improve the functionality of any affinity-type bioreactor. Indeed, efforts have been made recently in utilizing these approaches to develop a clinically usable GPIIb/IIIa bioreactor for the treatment of immune thrombocytopenic purpura (ITP)-an autoimmune disease.

Anticoagulants↗

PubMed2005

The minimal functional sequence of protamine.

Despite its nearly universal applications, protamine, a mixture of four major peptides with different sequences, is associated with clinically significant side effects. Through a well-designed enzyme digestion method, various low molecular weight protamine peptides were obtained. Among them, two low molecular weight protamine peptides with the same or even more potent heparin neutralization abilities as native protamine were identified through both in vitro and in vivo tests. In addition, in vivo experiments showed that compared to native protamine, these two low molecular weight protamine peptides were less toxic and would be safer for clinical use.

Amino Acid Sequence↗

PubMed2005

Insulin-cell penetrating peptide hybrids with improved intestinal absorption efficiency.

Cell-penetrating peptide (CPP) was linked to insulin to form insulin-CPP hybrids. The intestinal absorption efficiency of CPP hybridized insulin was 6-8 times increased compared to normal insulin as tested on Caco-2 cell monolayer, a widely used in vitro model for intestinal absorption. Insulin-CPP hybrid transportation seemed to be through an active and transcytosis-like mechanism. Importantly, insulin in hybrids kept intact after they passed through the Caco-2 cell monolayer. This study provides a new clue for oral insulin development.

Administration, Oral↗

PubMed2005

Nontoxic membrane translocation peptide from protamine, low molecular weight protamine (LMWP), for enhanced intracellular protein delivery: in vitro and in vivo study.

Naturally derived, nontoxic peptides from protamine by the authors, termed low molecular weight protamines (LMWPs), possess high arginine content and carry significant sequence similarity to that of TAT, by far the most potent protein transduction domain peptide. Therefore, it was hypothesized that these LMWPs would also inherit the similar translocation activity across the cell membrane, which enables any impermeable species to be transduced into the cells. LMWPs were prepared by enzymatic digestion of protamine, examined their capability of transducing an impermeable protein toxin into the tumor cells by chemical conjugation, and determined cytotoxicity of transduced protein toxin (e.g., gelonin) against cancer cell lines and a tumor-bearing mouse. In vitro results showed that LMWPs could indeed translocate themselves into several mammalian cell lines as efficiently as TAT, thereby transducing impermeable gelonin into the cells by chemical conjugation. In vivo studies further confirmed that LMWP could carry an impermeable gelonin across the tumor mass and subsequently inhibit the tumor growth. In conclusion, the presence of equivalent cell translocation potency, absence of toxicity of peptide itself, and the suitability for low-cost production by simple enzymatic digestion could expand the range of clinical applications of LMWPs, including medical imaging and gene/protein therapies.

Animals↗

PubMed2005

Application of "ATTEMPTS" for drug delivery.

A novel enzyme drug delivery system, Antibody, Targeted, Triggered, Electrically, Modified, Prodrug, Type, Strategy ("ATTEMPTS"), was developed in our laboratory to attenuate the toxicity associated with drug activity at non-targeted tissues. Tissue plasminogen activator is a prime example of an enzyme drug that exhibits systemic toxicity due to its indiscriminate activation of both targeted (i.e., clot-bound) and non-targeted (i.e., systemic) plasminogen. In brief, tissue plasminogen activator (t-PA) was modified to contain positive surface charges and then rendered inactive upon electrostatic binding with a negatively charged heparin-antifibrin antibody conjugate. After targeting the complex to the clot site, t-PA activity was restored by administration of protamine, a clinical heparin antidote. Cation-modified t-PA (CM-tPA) was obtained by chemical conjugation of t-PA with a poly(Arg)7Cys peptide using the crosslinker N-succinimidyl 3-2-(pyridlydithio)propionate (SPDP). Anti-fibrin IgG was chemically conjugated with heparin via oxidation of the carbohydrate moiety on its Fc region. Both in vitro characterization and in vivo studies using a rat thrombosis model clearly demonstrated that heparin-IgG conjugate induced inhibition of CM-tPA could be effectively reversed upon addition of protamine. Overall, the ATTEMPTS system was proven to induce clot dissolution without causing t-PA associated systemic toxicity due to the degradation of critical plasma factors by systemic plasmin production.

Animals↗

PubMed2004

Injectable microparticle-gel system for prolonged and localized lidocaine release. I. In vitro characterization.

Current treatment protocol for postoperative pain is to infuse anesthetic solution around nerves or into the epidural space. This clinical practice is beset by the short duration of the anesthetic effect unless the infusion is continuous. Continuous infusion, however, requires hospitalization of the patients, thereby increasing medical costs. In addition, it also causes systemic accumulation of the drug. We reported herein a novel treatment for the postoperative pain by applying to the surgical site a biodegradable microsphere-gel system for prolonged and localized release of encapsulated anesthetic drugs. This lidocaine-containing biodegradable poly(D,L-lactic acid) (PLA) microsphere system, although being established previously by other investigators, was hindered by a burst release and a followed rapid release of the drug within several hours in vitro. In this article, we demonstrated that by a step-by-step modification of the formulation, prolonged release of lidocaine, up to several days in vitro, could be achieved. Differential scanning calorimetry revealed a lower glass transition temperature for these lidocaine-loaded microspheres comparing to that of lidocaine-free microspheres. This decreased Tg explained for the tendency of the lidocaine-loaded microspheres to physically fuse at higher temperatures. In vitro studies showed that microspheres, when loaded with 35% lidocaine, yielded a threefold increase in the degradation rate. The molecular weight of PLA of the drug-loaded microspheres was reduced by 50% within a period of 1 month. Based on the results (of prolonged lidocaine release and rapid PLA microsphere degradation), this lidocaine-loaded PLA microsphere system could offer a simple solution to the treatment of postoperative pain.

Anesthetics, Local↗

PubMed2004

Injectable microparticle-gel system for prolonged and localized lidocaine release. II. In vivo anesthetic effects.

Current treatment protocols for postoperative pain are beset by either the short duration of the anesthetic effect or requirement of hospitalization of the patients. We reported herein a novel treatment by applying to the surgical site a biodegradable microparticle-gel system for prolonged and localized release of encapsulated anesthetic drugs. In a previous publication, lidocaine-loaded poly(D,L-lactic acid) microspheres were fabricated and their formulations were optimized. In vitro characterization of these lidocaine-loaded microspheres, however, revealed a shortcoming of this system; that is, microspheres tend to fuse physically. Fusion of the microspheres could hinder their clinical applications, as it would clog the needle. In this article, we demonstrated that fabricating microspheres with high molecular weight (approximately 60 KDa) poly(lactic-co-glycolic acid) would increase the glass transition temperature of the microspheres after lidocaine loading, thereby increasing their mechanical stability and eliminating their fusion during storage. Such microspheres containing 31% (w/w) lidocaine in the presence or absence of 25% (w/v) poloxamer 407 gel were then evaluated in vivo by monitoring the sensory and motor functions of the rats after sciatic nerve block, using the previously established hot-plate and weight-bearing testing methods. Results showed that microspheres formulated with poloxamer 407 gel yielded the longest duration of sensory and motor block for a period of approximately 8.5 h, compared to 5 h by microspheres in saline, 5 h by lidocaine in poloxamer 407 gel, and 2 h by lidocaine in saline. This study suggests that the microsphere-gel system containing lidocaine could potentially be applied clinically to the treatment of postoperative pain.

Anesthetics, Local↗

PubMed2004

Development of protamine hollow-fiber device for extracorporeal heparin removal.

Protamine was covalently coupled to a cellulose hollow fiber (cuprophan membrane) through epichlorohydrin-activated hydroxyl groups on the hollow fiber. An immobilization efficiency of as much as 15 mg protamine/g fiber could be achieved under optimal reacting conditions. Compared to a protamine hollow-fiber device produced using cyanogen bromide as an activator, the protamine hollow-fiber device using epichlorohydrin as an activator shows very high resistance to hydrolysis. Both in vitro and in vivo experiments showed that such a protamine hollow-fiber device had high heparin-removal capacity and efficiency, suggesting that this device may be suitable for various clinical applications in extracorporeal heparin removal.

Adsorption↗

PubMed2003

Surface-modified poly(lactide-co-glycolide) nanospheres for targeted bone imaging with enhanced labeling and delivery of radioisotope.

Surface-modified nanospheres can be utilized for targeting drugs and diagnostic agents to the bone and bone marrow while extending their circulation time in the blood stream. The surface modification of poly(lactide-co-glycolide) (PLGA) nanospheres by radioisotope carrying poly(ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) triblock copolymers (Poloxamer 407) has been assessed by in vitro characterization and in vivo biodistribution studies after intravenous administration of the nanospheres to the mouse. A hydroxyphenylpropionic acid, a ligand for (125)I and (131)I labeling, was conjugated to the hydroxyl group of the Poloxamer 407 by using dicyclohexyl carbodiimide. The ligand-conjugated Poloxamer 407 was adsorbed onto the surface of PLGA nanospheres. Surface coating was confirmed by measuring both size distribution and the surface charge of the nanospheres. Besides, (125)I-labeling efficiency, radiolabeling stability, whole body imaging, and biodistribution of the radioisotope-labeled nanospheres were examined. Ligand-labeled, surface-modified PLGA nanospheres were in 100-nm size ranges, which may be adequate for long-circulation and further bone imaging. (125)I-labeling efficiency was >90% and was more stable at human serum for 24 h. A noticeable decrease in liver or spleen uptake was obtained by the surface-modified nanospheres. (125)I-labeled nanospheres showed higher blood maintenance and bone uptake compared with stannous colloid with the same size distribution. Therefore, a fully biodegradable, radioisotope-carrying, surface-modified nanosphere system has been developed as a promising tool for targeting bone and bone marrows.

Animals↗

PubMed2003

ATTEMPTS: a heparin/protamine-based triggered release system for the delivery of enzyme drugs without associated side-effects.

A prodrug type delivery system based on competitive ionic binding for the conversion of the prodrug to an active drug has been developed for delivery of enzyme drugs without their associated toxic side-effects. This approach, termed "ATTEMPTS" (antibody targeted, triggered, electrically modified prodrug-type strategy), would permit the administration of an inactive drug and then subsequently triggered release of the active drug at the target site. The underlying principle was to modify the enzyme with small cationic species so that it could bind a negatively charged heparin-linked antibody, and the latter would block the activity of the enzyme drug until it reached the target. To provide the enzyme drug with appropriate binding strength to heparin, a cationic poly(Arg)(7) peptide was incorporated onto the enzyme either by the chemical conjugation method using a bifunctional crosslinker or by the biological conjugation method using the recombinant methodology. Methods for drug modification, heparin-antibody conjugation, and the prodrug and triggered release features of the "ATTEMPTS" approach are described in detail in this review article.

Antibodies↗

PubMed2003

Low molecular weight protamine as an efficient and nontoxic gene carrier: in vitro study.

BACKGROUND: The structural similarity between low molecular weight protamine (LMWP), prepared by enzymatic digestion of protamine, and HIV-TAT protein transduction peptide suggested the feasibility of LMWP as an efficient carrier for delivering therapeutic genes while alleviating the cytotoxicity of currently employed gene carriers. METHODS: LMWP was prepared by enzymatic digestion of protamine with thermolysine. The prepared LMWP peptide and TAT peptide, as well as their complexes with plasmid DNA (pDNA), were examined for cellular uptake behaviors by using confocal microscopy and flow cytometry. The complexation of pDNA and LMWP was monitored by gel retardation test as well as size and zeta potential measurements, and was then further assessed by DNase I protection assay. The transfection efficiency of pDNA/LMWP was examined by varying the pDNA content and charge ratio in the complex, and then compared with that of pDNA/PEI. Cytotoxicity induced by pDNA/LMWP and pDNA/PEI was also examined. RESULTS: Prepared LMWP showed similar transcellular localization behavior and kinetics to those of TAT, and efficiently transferred the pDNA into nucleus and cytoplasm in a short time period. The size and zeta potential of the pDNA/LMWP complex were 120 nm and 30 mV, respectively, which were adequately suitable for cellular uptake. After forming the complex, LMWP appeared to effectively protect pDNA against DNase I attack. The pDNA/LMWP complex showed significantly enhanced gene transfer than both naked pDNA and the pDNA/PEI complex, while exhibiting a markedly reduced cytotoxicity than that of the pDNA/PEI complex. CONCLUSIONS: The present study suggested that LMWP could be a useful and safe tool for enhancing delivery of bioactive molecules and therapeutic DNA products into cells when applied in gene therapy.

Amino Acid Sequence↗

PubMed2002

Poly(glutamic acid) poly(ethylene glycol) hydrogels prepared by photoinduced polymerization: Synthesis, characterization, and preliminary release studies of protein drugs.

A class of new biodegradable hydrogels based on poly(ethylene glycol) methacrylate-graft-poly(glutamic acid) and poly(ethylene glycol) dimethacrylate was synthesized by photoinduced polymerization. Because all the polymeric constituents were highly hydrophilic, crosslinking could be performed in aqueous solutions. This type of crosslinked hydrogel was prepared by modifying a select number of acidic side-groups on poly(glutamic acid) with poly(ethylene glycol) methacrylate. These modified chains were then crosslinked in the presence of poly(ethylene glycol) dimethacrylate under a photoinduced polymerization at a wavelength of 365 nm. Swelling experiments were conducted to study the crosslinking density, pH-responsive behavior, and degradation of the hydrogel. Results showed that the degree of swelling of this type of hydrogels increased as the crosslinker concentration (or density) was reduced. Because of the presence of acidic side chains on poly(glutamic acid), swelling behavior was found to be pH-responsive, increasing at high pH in response to the increase in the amount of ionized acidic side chains. The degradation rate of these hydrogels also varied with pH. More rapid degradation was observed under stronger alkaline conditions because of the hydrolysis of the ester bonds between the crosslinker and the polymer backbone. Practically useful degradation rates could be achieved for such hydrogels under physiological conditions. Drug release rates from these hydrogels were found to be proportional to the protein molecular weight and the crosslinker density; increasing at lower protein molecular weight or crosslinker density. The preliminary findings presented in this article suggest that this class of biodegradable hydrogels could be an attractive avenue for drug delivery applications. The specific photoinduced crosslinking chemistry used would permit hydrogels to be synthesized in existence of the entrapped macromolecular drugs including peptides, proteins, and cells. In addition, the rapid feature of this polymerization procedure along with the ability to perform hydrogel synthesis and drug loading in an aqueous environment would offer great advantages in retaining drug activity during hydrogel synthesis.

Biocompatible Materials↗

PubMedDate not supplied

PEG-modified protamine with improved pharmacological/pharmaceutical properties as a potential protamine substitute: synthesis and in vitro evaluation.

Cardiopulmonary bypass (CPB) procedures are frequently associated with massive inflammatory responses, resulting in a high rate of morbidity and mortality in routine cardiac operations. One recognized attribute of these deleterious responses is the synergic effect of heparin and protamine, which elicit the activation of the complement system in vivo. To circumvent such toxic effects following protamine reversal of heparin anticoagulation in the CPB procedures, we proposed that poly(ethylene glycol) (PEG)-modified protamine could retain the heparin-neutralization ability and yet diminish the induced complement activation by the formed heparin-protamine complexes (HPC), thereby providing highly improved pharmacological properties. PEGylation of protamine was carried out by utilizing N-hydroxysuccinimidyl (NHS) conjugation chemistry. Size exclusion chromatography (SEC), reverse-phase high performance liquid chromatography (RP-HPLC), and matrix-assisted laser desorption mass spectrometry (MALDI-MS) were used to assess the conjugation stiochiometry, the purity of the conjugates, and the site of PEG modification, respectively. The heparin-neutralizing activity was determined by using heparin affinity chromatography and various biological assays including the plasma-activated partial thromboplastin time (aPTT), anti-Xa, and anti-IIa methods. The potency in inducing complement activation was examined in vitro using the CH50 hemolytic assay. The PEG-modified protamine was successfully synthesized with a PEG/protamine stiochiometry of 1:1. Only one conjugation site for PEG that was located at the N-terminal end of protamine was obtained. In the biological evaluations, the PEG-modified protamine displayed a full retention of the heparin-neutralizing ability of protamine and a significantly reduced activity in complement activation following its complexation with heparin. Results from studies of the particle size and zeta potential indicated that the PEG-modified protamine formed substantially smaller aggregates with heparin, rendering them less effective in triggering the size-dependent complement responses. As with protamine, PEG-modified protamine exhibited an enhanced aqueous solubility, therefore attaining significantly improved pharmaceutical properties. These preliminary results suggested that the PEG-modified protamine conjugate might serve as a potential protamine substitute with improved therapeutic and pharmaceutical properties in heparin reversal.

Blood Coagulation↗