Shape of things to come in platelet mimicry

By mimicking the shape, size, flexibility and surface chemistry of real platelets, artificial platelets are pushed out of the main blood flow to vessel walls. There, the surface chemistry enables them to anchor on damaged cells and induce faster clotting at the site. Credit: Anirban Sen Gupta

Artificial platelet mimics developed by a research team from Case Western Reserve University and University of California, Santa Barbara, are able to halt bleeding in mouse models 65 percent faster than nature can on its own.

For the first time, the researchers have been able to integratively mimic the shape, size, flexibility and surface chemistry of real blood platelets on albumin-based particle platforms. The researchers believe these four design factors together are important in inducing clots to form faster selectively at vascular injury sites while preventing harmful clots from forming indiscriminately elsewhere in the body.

The new technology, reported in the journal ACS Nano, is aimed at stemming bleeding in patients suffering from traumatic injury, undergoing surgeries or suffering clotting disorders from platelet defects or a lack of platelets. Further, the technology may be used to deliver drugs to target sites in patients suffering atherosclerosis, thrombosis or other platelet-involved pathologic conditions.

Anirban Sen Gupta, an associate professor of biomedical engineering at Case Western Reserve, previously designed peptide-based surface chemistries that mimic the clot-relevant activities of real platelets. Building on this work, Sen Gupta now focuses on incorporating morphological and mechanical cues that are naturally present in platelets to further refine the design.

"Morphological and mechanical factors influence the margination of natural platelets to the blood vessel wall, and only when they are near the wall can the critical clot-promoting chemical interactions take place," he said.

These natural cues motivated Sen Gupta to team up with Samir Mitragotri, a professor of chemical engineering at UC Santa Barbara, whose laboratory has recently developed albumin-based technologies to make particles that mimic the geometry and mechanical properties of red blood cells and platelets.

Together, the team has developed artificial platelet-like nanoparticles (PLNs) that combine morphological, mechanical and surface chemical properties of natural platelets.

The researchers believe this refined design will be able to simulate natural platelet's ability to collide effectively with larger and softer red blood cells in systemic blood flow. The collisions cause margination -- pushing the platelets out of the main flow and closer to the blood vessel wall -- increasing the probability of interacting with an injury site.

The surface coatings enable the artificial platelets to anchor to injury-site-specific proteins, von Willebrand Factor and collagen, while inducing the natural and artificial platelets to aggregate faster at the injury site.

Testing in mouse models showed that intravenous injection of these artificial platelets formed clots at the site of injury three times faster than natural platelets alone in control mice.

The ability to interact selectively with injury site proteins, as well as the ability to remain mechanically flexible like natural platelets, enables these artificial platelets to safely ride through the smallest of blood vessels without causing unwanted clots.

Albumin, a protein found in blood serum and eggs, is already used with cancer drugs and considered a safe material. Artificial platelets that don't become involved in a clot and continue to circulate are metabolized within one to two days.

The researchers believe the new artificial platelet design may be even more effective in larger volume blood flows where margination to the blood vessel wall is more prominent. They expect to soon begin testing those capabilities.

This research was previously funded by American Heart Association and is currently funded by National Institutes of Health.

In addition to stemming bleeding, Sen Gupta believes the technology could also be useful in delivering clot-busting medicines directly to clots, to treat heart attack or stroke without having to systemically suspend the body's coagulation mechanism. The artificial platelets may also be used to deliver cancer medicines to metastatic tumors that have high platelet interactions. Sen Gupta is seeking grants to pursue that work.

Source: Case Western Reserve University

Bio-inspired bleeding control: Synthesized platelet-like nanoparticles created

Artist's rendering of synthetic platelets. Credit: Peter Allen illustration

Stanching the free flow of blood from an injury remains a holy grail of clinical medicine. Controlling blood flow is a primary concern and first line of defense for patients and medical staff in many situations, from traumatic injury to illness to surgery. If control is not established within the first few minutes of a hemorrhage, further treatment and healing are impossible.

At UC Santa Barbara, researchers in the Department of Chemical Engineering and at Center for Bioengineering (CBE) have turned to the human body's own mechanisms for inspiration in dealing with the necessary and complicated process of coagulation. By creating nanoparticles that mimic the shape, flexibility and surface biology of the body's own platelets, they are able to accelerate natural healing processes while opening the door to therapies and treatments that can be customized to specific patient needs.

"This is a significant milestone in the development of synthetic platelets, as well as in targeted drug delivery," said Samir Mitragotri, CBE director, who specializes in targeted therapy technologies. Results of the researchers' findings appear in the current issue of the journal ACS Nano.

The process of coagulation is familiar to anyone who has suffered even the most minor of injuries, such as a scrape or paper cut. Blood rushes to the site of the injury, and within minutes the flow stops as a plug forms at the site. The tissue beneath and around the plug works to knit itself back together and eventually the plug disappears.

But what we don't see is the coagulation cascade, the series of signals and other factors that promote the clotting of blood and enable the transition between a free-flowing fluid at the site and a viscous substance that brings healing factors to the injury. Coagulation is actually a choreography of various substances, among the most important of which are platelets, the blood component that accumulates at the site of the wound to form the initial plug.

"While these platelets flow in our blood, they're relatively inert," said graduate student researcher Aaron Anselmo, lead author of the paper. As soon as an injury occurs, however, the platelets, because of the physics of their shape and their response to chemical stimuli, move from the main flow to the side of the blood vessel wall and congregate, binding to the site of the injury and to each other. As they do so, the platelets release chemicals that "call" other platelets to the site, eventually plugging the wound.

But what happens when the injury is too severe, or the patient is on anti-coagulation medication, or is otherwise impaired in his or her ability to form a clot, even for a modest or minor injury?

That's where platelet-like nanoparticles (PLNs) come in. These tiny, platelet-shaped 

particles that behave just like their human counterparts can be added to the blood flow to supply or augment the patient's own natural platelet supply, stemming the flow of blood and initiating the healing process, while allowing physicians and other caregivers to begin or continue the necessary treatment. Emergency situations can be brought under control faster, injuries can heal more quickly and patients can recover with fewer complications.

"We were actually able to render a 65 percent decrease in bleeding time compared to no treatment," said Anselmo.

According to Mitragotri, the key lies in the PLNs' mimicry of the real thing. By imitating the shape and flexibility of natural platelets, PLNs can also flow to the injury site and congregate there. With surfaces functionalized with the same biochemical motifs found in their human counterparts, these PLNs also can summon other platelets to the site and bind to them, increasing the chances of forming that essential plug. In addition, and very importantly, these platelets are engineered to dissolve into the blood after their usefulness has run out. This minimizes complications that can arise from emergency hemostatic procedures.

"The thing about hemostatic agents is that you have to intervene to the right extent," said Mitragotri. "If you do too much, you cause problems. If you do too little, you cause problems."

These synthetic platelets also let the researchers improve on nature. According to Anselmo's investigations, for the same surface properties and shape, nanoscale particles can perform even better than micron-size platelets. Additionally, this technology allows for customization of the particles with other therapeutic substances -- medications, therapies and such -- that patients with specific conditions might need.

"This technology could address a plethora of clinical challenges," said Dr. Scott Hammond, director of UCSB's Translational Medicine Research Laboratories. "One of the biggest challenges in clinical medicine right now -- which also costs a lot of money -- is that we're living longer and people are more likely to end up on blood thinners. When an elderly patient presents at a clinic, it's a huge challenge because you have no idea what their history is and you might need an intervention."

With optimizable PLNs, physicians would be able to strike a finer balance between anticoagulant therapy and wound healing in older patients, by using nanoparticles that can target where clots are forming without triggering unwanted bleeding. In other applications, bloodborne pathogens and other infectious agents could be minimized with antibiotic-carrying nanoparticles. Particles could be made to fulfill certain requirements to travel to certain parts of the body -- across the blood-brain barrier, for instance -- for better diagnostics and truly targeted therapies.

Additionally, according to the researchers, these synthetic platelets cost relatively less, and have a longer shelf life than do human platelets -- a benefit in times of widespread emergency or disaster, when the need for these blood components is at its highest and the ability to store them onsite is essential.

Further research into PLNs will involve investigations to see how well the technology and synthesis can scale up, as well as assessments into the more practical matters involved in translating the technology from the lab to the clinic, such as manufacturing, storage, sterility and stability as well as pre-clinical and clinical testing.

Source: University of California - Santa Barbara

Nanotechnology against malaria parasites

After maturation, malaria parasites (yellow) are leaving an infected red blood cell and are efficiently blocked by nanomimics (blue). Credit: Fig: modified by University of Basel with permission from ACS

Malaria parasites invade human red blood cells, they then disrupt them and infect others. Researchers at the University of Basel and the Swiss Tropical and Public Health Institute have now developed so-called nanomimics of host cell membranes that trick the parasites. This could lead to novel treatment and vaccination strategies in the fight against malaria and other infectious diseases. Their research results have been published in the scientific journal ACS Nano.

For many infectious diseases no vaccine currently exists. In addition, resistance against currently used drugs is spreading rapidly. To fight these diseases, innovative strategies using new mechanisms of action are needed. The malaria parasite Plasmodium falciparum that is transmitted by the Anopheles mosquito is such an example. Malaria is still responsible for more than 600,000 deaths annually, especially affecting children in Africa (WHO, 2012).
Artificial bubbles with receptors

Malaria parasites normally invade human red blood cells in which they hide and reproduce. They then make the host cell burst and infect new cells. Using nanomimics, this cycle can now be effectively disrupted: The egressing parasites now bind to the nanomimics instead of the red blood cells.

Researchers of groups led by Prof. Wolfgang Meier, Prof. Cornelia Palivan (both at the University of Basel) and Prof. Hans-Peter Beck (Swiss TPH) have successfully designed and tested host cell nanomimics. For this, they developed a simple procedure to produce polymer vesicles -- small artificial bubbles -- with host cell receptors on the surface. The preparation of such polymer vesicles with water-soluble host receptors was done by using a mixture of two different block copolymers. In aqueous solution, the nanomimics spontaneously form by self-assembly.

Blocking parasites efficiently

Usually, the malaria parasites destroy their host cells after 48 hours and then infect new red blood cells. At this stage, they have to bind specific host cell receptors. Nanomimics are now able to bind the egressing parasites, thus blocking the invasion of new cells. The parasites are no longer able to invade host cells, however, they are fully accessible to the immune system.

The researchers examined the interaction of nanomimics with malaria parasites in detail by using fluorescence and electron microscopy. A large number of nanomimics were able to bind to the parasites and the reduction of infection through the nanomimics was 100-fold higher when compared to a soluble form of the host cell receptors. In other words: In order to block all parasites, a 100 times higher concentration of soluble host cell receptors is needed, than when the receptors are presented on the surface of nanomimics.

"Our results could lead to new alternative treatment and vaccines strategies in the future," says Adrian Najer first-author of the study. Since many other pathogens use the same host cell receptor for invasion, the nanomimics might also be used against other infectious diseases. The research project was funded by the Swiss National Science Foundation and the NCCR "Molecular Systems Engineering."

Source: University of Basel

Promising compound rapidly eliminates malaria parasite

A new report says that the rapid action of (+)-SJ733 will likely slow malaria drug resistance. Credit: Peter Barta, St. Jude Children's Research Hospital

An international research collaborative has determined that a promising anti-malarial compound tricks the immune system to rapidly destroy red blood cells infected with the malaria parasite but leave healthy cells unharmed. St. Jude Children's Research Hospital scientists led the study, which appears in the current online early edition of the Proceedings of the National Academy of Sciences (PNAS).

The compound, (+)-SJ733, was developed from a molecule identified in a previous St. Jude-led study that helped to jumpstart worldwide anti-malarial drug development efforts. Malaria is caused by a parasite spread through the bite of an infected mosquito. The disease remains a major health threat to more than half the world's population, particularly children. The World Health Organization estimates that in Africa a child dies of malaria every minute.

In this study, researchers determined that (+)-SJ733 uses a novel mechanism to kill the parasite by recruiting the immune system to eliminate malaria-infected red blood cells. In a mouse model of malaria, a single dose of (+)-SJ733 killed 80 percent of malaria parasites within 24 hours. After 48 hours the parasite was undetectable.

Planning has begun for safety trials of the compound in healthy adults.

Laboratory evidence suggests that the compound's speed and mode of action work together to slow and suppress development of drug-resistant parasites. Drug resistance has long undermined efforts to treat and block malaria transmission.

"Our goal is to develop an affordable, fast-acting combination therapy that cures malaria with a single dose," said corresponding author R. Kiplin Guy, Ph.D., chair of the St. Jude Department of Chemical Biology and Therapeutics. "These results indicate that SJ733 and other compounds that act in a similar fashion are highly attractive additions to the global malaria eradication campaign, which would mean so much for the world's children, who are central to the mission of St. Jude."

Whole genome sequencing of the Plasmodium falciparum, the deadliest of the malaria parasites, revealed that (+)-SJ733 disrupted activity of the ATP4 protein in the parasites. The protein functions as a pump that the parasites depend on to maintain the proper sodium balance by removing excess sodium.

The sequencing effort was led by co-author Joseph DeRisi, Ph.D., a Howard Hughes Medical Institute investigator and chair of the University of California, San Francisco Department of Biochemistry and Biophysics. Investigators used the laboratory technique to determine the makeup of the DNA molecule in different strains of the malaria parasite.

Researchers showed that inhibiting ATP4 triggered a series of changes in malaria-infected red blood cells that marked them for destruction by the immune system. The infected cells changed shape and shrank in size. They also became more rigid and exhibited other alterations typical of aging red blood cells. The immune system responded using the same mechanism the body relies on to rid itself of aging red blood cells.

Another promising class of antimalarial compounds triggered the same changes in red blood cells infected with the malaria parasite, researchers reported. The drugs, called spiroindolones, also target the ATP4 protein. The drugs include NITD246, which is already in clinical trials for treatment of malaria. Those trials involve investigators at other institutions.

"The data suggest that compounds targeting ATP4 induce physical changes in the infected red blood cells that allow the immune system or erythrocyte quality control mechanisms to recognize and rapidly eliminate infected cells," DeRisi said. "This rapid clearance response depends on the presence of both the parasite and the investigational drug. That is important because it leaves uninfected red blood cells, also known as erythrocytes, unharmed."

Laboratory evidence also suggests that the mechanism will slow and suppress development of drug-resistant strains of the parasite, researchers said.

Planning has begun to move (+)-SJ733 from the laboratory into the clinic beginning with a safety study of the drug in healthy adults. The drug development effort is being led by a consortium that includes scientists at St. Jude, the Swiss-based non-profit Medicines for Malaria Venture and Eisai Co., a Japanese pharmaceutical company.

Source: St. Jude Children's Research Hospital