Polymer-peptide conjugate called HAPPI can reduce 99 percent in bleeding time and a 97 percent reduction in blood loss

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When it comes to traumatic injuries, it’s a race against time.

A person with major hemorrhage can die from blood loss within minutes, so staunching the wound and getting them to a hospital as fast as possible is critical.

Bleeding from the extremities can be slowed with compression but what about internal bleeding?

In a hospital, internal bleeding can be controlled with the transfusion of clotting agents, such as platelets, but they require careful storage and refrigeration and can’t be carried by first responders.

As a result, the majority of people who succumb to traumatic injuries outside a hospital die from treatable hemorrhages.

Now, researchers from the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS), in collaboration with Massachusetts General Hospital, Beth Israel Deaconess Medical Center, and Case Western Reserve University, report an injectable clotting agent that reduced blood loss by 97 percent in mice models.

The freeze-dried agent, which has a physical consistency of cotton candy, can be stored at room temperature for several months and reconstituted in saline before injection.

The research is published in Science Advances.

“Our goal was to give first responders a tool to stop internal bleeding that could be easily carried in a backpack or stored in an ambulance and, once injected intravenously in hemorrhagic patients, stop internal bleeding for a period long enough to get the patient to a hospital,” said Samir Mitragotri, Hiller Professor of Bioengineering and Hansjorg Wyss Professor of Biologically Inspired Engineering at SEAS and senior author of the study.

Mitragotri is also a Core Faculty member at Harvard’s Wyss Institute for Biologically Inspired Engineering.

Mitragotri and his team developed a polymer-peptide conjugate called HAPPI (Hemostatic Agents via Polymer Peptide Interfusion) that can selectively bind to damaged blood vessels and activated platelets at the bleeding site.

Circulating platelets are like the body’s EMTs – they are constantly surveying the body for wounds. When there is an injury to a blood vessel, the platelets get activated and attach themselves to the damaged vessel, causing a blood clot.

HAPPI binds to these activated platelets and enhances their accumulation at a bleeding site. It can be injected anywhere in the body and still make its way to the wound.

In mice models, HAPPI significantly lowered the bleeding time and bleeding volume of injuries. The researchers observed about a 99 percent reduction in bleeding time and a 97 percent reduction in blood loss.

The researchers also found that for traumatic injuries, the injection of HAPPI increased the median survival rate beyond one hour – a critical goal for trauma care.

“A lot of trauma-related deaths happen within the first hour when blood loss is happening profusely and there is no intervention,” said Yongsheng Gao, a postdoctoral research associate at SEAS and the co-first author of the paper.

“A key objective for first responders is to keep trauma patients alive during this so-called golden hour and in that time bring them to a hospital because once they get to the hospital, it’s a different game altogether.”

“With HAPPI, we sought to develop a safe and effective internal bandage,” said Apoorva Sarode, a former graduate student at SEAS and the co-first author of the study.

“We think that the simple design and scalable synthesis process of HAPPI will facilitate its seamless scale-up and translation to larger animal models, and eventually to the patients.”

Funding from Harvard’s Blavatnik Biomedical Accelerator enabled the lab to advance and validate the technology in animal models. Going forward, the team aims to scale up the production of the materials and test it in larger animal models..

Harvard’s Office of Technology Development has protected the intellectual property associated with this project and is exploring commercialization opportunities.

The paper was co-authored by Anvay Ukidve and Zongmin Zhao from Harvard SEAS, Shihui Guo and Robert Flaumenhaft from Beth Israel Deaconess Medical Center, Anirban Sen Gupta from Case Western Reserve University, and Nikolaos Kokoroskos and Noelle Saillant from Massachusetts General Hospital.


Chemical and structural characterization of HAPPI
HAPPI was characterized by nuclear magnetic resonance (NMR) spectroscopy, GPC, and atomic force microscopy (AFM) to confirm the successful conjugation and to probe their morphology. Characteristic 1H NMR peaks from VBP, CBP, and HA were detected in the final, purified products, demonstrating the successful conjugation (Fig. 1B).

Specifically, the peaks at 6.8 and 7.1 parts per million (ppm) correspond to aromatic rings in tyrosine from VBP. The peaks at 8.6 and 7.3 ppm belong to histidine from VBP and CBP, respectively.

In addition, the peak at 1.95 to 2.05 ppm corresponds to methyl groups in the acetamido moiety of HA. For a quantitative analysis, the methyl resonance of acetamido moiety of HA at (δ 1.95 to 2.05 ppm) was used as an internal standard (a in Fig. 1B).

The degree of VBP modification was estimated as ca. 7.8 mole percent (mol %) by comparing the peak areas of tyrosine and HA (d and e in Fig. 1B). Similarly, the CBP modification was found to be ~5.6 mol %.

On the basis of this, the average numbers of conjugated VBP and CBP molecules per single HA chain were estimated to be 51 and 37, respectively. VBP and CBP were also conjugated to HA individually in a similar manner, yielding HA-VBP and HA-CBP conjugates with comparable degree of substitution achieved (fig. S2).

Successful conjugation to HA was further confirmed by GPC (Fig. 1C, fig. S1, B and C). Unmodified HA does not absorb at 280 nm, and therefore, no peak was observed in ultraviolet (UV) detection.

On the other hand, HAPPI absorbs at the same location and exhibits the refractive index (RI) peak, indicating that the peptides were covalently attached to the HA molecules and are not just physically mixed with the polymer. A

similar retention volume of AF 647–HA and AF 647–HAPPI demonstrated that HA was intact, with no detectable degradation occurring during the conjugation reaction and purification process.

Fig. 1 Synthesis and characterization of AF 647–labeled HA-CBP-VBP conjugates (HAPPI).(A) Reaction scheme for conjugation of fluorescent dye and peptides on HA backbone using EDC/sulfo-NHS chemistry. (B1H nuclear magnetic resonance (NMR) spectrum shows the presence of characteristic peaks b, c, d, and e from the peptides, indicating their successful conjugation to HA. (C) GPC profile of HAPPI shows the appearance of ultraviolet (UV) absorbance at the elution time of conjugates [refractive index (RI) signals], in contrast to the profile of fluorescent HA alone, further validating the chemical conjugation. (D) Image of freeze-dried HAPPI as the bluish material in the vial. Topographic air tapping mode atomic force micrographs for HA (E), HA-VBP (F), HA-CBP (G), and HAPPI (H) (scale bars, 500 nm). Photo credit: (D) Y. Gao, Harvard University.

The increase in molecular weights after conjugation (HA-VBP, HA-CBP, and HAPPI versus HA), as determined from their nonfluorescent analogs, is further manifested by the parallel shift of molecular weight distribution peaks (fig. S1, D and E).

To assess the storage stability of HAPPI, they were stored at room temperature in lyophilized solids (Fig. 1D) for 3 months and assessed by GPC. The elution profile of the conjugates was comparable to the starting materials, demonstrating the outstanding stability of these formulations.

To probe their morphology, HA and HAPPI, along with conjugates of HA with individual peptides, were deposited on a highly ordered pyrolytic graphite (HOPG) substrate from dilute aqueous solutions and imaged in air by tapping mode AFM (Fig. 1, E to H).

HA molecules exhibited both condensed globular forms and extended coil structures, which are known to be due to the intramolecular and intermolecular aggregation (24).

While both globular and coiled forms were also observed for HA-VBP, HA-CBP, and HAPPI, HA-VBP tends to form extended conformation, whereas HA-CBP forms more isolated globular forms.

This conformational difference could be attributed to the difference in hydrophobicity and isoelectric points of VBP and CBP, which can regulate their molecular conformations both in the solution and on the surface (24).

While it would be ideal to characterize the morphology of HAPPI in physiologically relevant condition, such as in saline solution, the hydrophilic nature of HA backbone makes it practically challenging, as HA has weaker affinity toward the substrate than to aqueous solution (24).

After modifying the mica substrate with positively charged polylysine, we obtained the morphology of HAPPI in saline (fig. S3), which is similar with those observed in air.

Reference

24. C. Spagnoli, A. Korniakov, A. Ulman, E. A. Balazs, Y. L. Lyubchenko, M. K. Cowman, Hyaluronan conformations on surfaces: Effect of surface charge and hydrophobicity. Carbohydr. Res. 340, 929–941 (2005).


More information: “A polymer-based systemic hemostatic agent” Science Advances (2020). advances.sciencemag.org/lookup … .1126/sciadv.aba0588

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