Researchers report on the development of camouflaging technique for drug-delivering nanoparticles that essentially cloaks the particles in red blood cell membranes. Liangfang Zhang, Ph.D., and colleagues at the Department of NanoEngineering and Moores Cancer Center, University of California, San Diego (UCSD) claim red blood cell (RBC) membrane-coated polymer nanoparticles evade the immune system, and can circulate for much longer in the body than existing polyethylene glycol-based nanoparticles. When injected into mice, the RBC membrane-camouflaged nanoparticles containing a fluorophore were still visible in the blood after 72 hours.
Funded by the National Institutes of Health and described in PNAS, the achievement could pave the way for cancer therapies based on drug-loaded nanoparticles coated in the patients’ own red blood cell membranes, the researchers suggest. As well as allowing chemotherapy to be administered using a single injection rather than requiring hours of intravenous infusion, the technology would be associated with an absolutely minimal risk of triggering an immune response. Dr. Zhang and colleagues' work is described in a paper titled “Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform.”
Different approaches to extending the circulaton time of nanoparticles in vivo include modifications on particle size, surface, shape, and flexibility, the researchers explain. The current gold standard for nanoparticle stealth coating is PEG, which has led to successes in a number of clinicial trials. However, the Moores team continues, work is also ongoing to try and extend the circulation time of nanoparticles even further by modeling nanocarriers on erythrocytes, which naturally circulate in the body for up to 180 days and act as “nature’s long-circulation delivery vehicle.”
One of the challenges in achieving this goal is associated with functionalizing nanoparticles with the complex surface chemistry of the cell. To address this, Dr. Zhang’s team looked at the problem from an engineering rather than a biological perspective. “If the red blood cell has such a feature and we know that it has something to do with the membrane—although we don’t fully understand exactly what is going on at the protein level—we just take the whole membrane,” Dr. Zhang notes. “You put the cloak on the nanoparticle, and the nanoparticle looks like a red blood cell.”
This is essentially what the researchers now claim to have achieved. The approach involved extruding sub-100 nm poly(lactic-co-glycolic) (PLGA) particles with preformed RBC-membrane-derived vesicles, to provide a coating of bilayered RBC membrane that included both lipids and surface proteins. The overall process was carried out in two stages: membrane vesicle derivation from RBCs, then vesicle-particle fusion.
Mouse RBCs were purified from fresh blood, and their membranes ruptured in a hypotonic environment to remove the intracellular contents. The empty RBCs were then washed and extruded through 100 nm porous membranes to create RBC-membrane-derived vesicles. These were then fused with roughly 70 nm-diameter PLGA nanoparticles using mechanical extrusion. The mechanical force facilitated the sub-100 nm PLGA nanoparticles to cross the lipid bilayers, resulting in vesicle-particle fusion. The authors claim repeated passages through the extruder negated potential issues with liposome-particle fusion, such as broad particle size distribution, incomplete particle coating, and inconsistent lipid shells.
Transmission electron microscopy of stained RBC membrane-coated nanoparticles showed that the structure comprised a polymeric core of about 70 nm in diameter and an outer lipid shell of 7–9 nm in thickness, matching the previously reported measurement for an RBC membrane. Subsequent comparative analysis of the protein composition of empty RBCs and RBC membrane-derived vesicles indicated that the membrane proteins were mostly retained throughout particle synthesis. “This finding suggests that the translocation of the bilayered cellular membranes also transfers the associated membrane proteins to the nanoparticle surface,” the authors write. “Because the solid PLGA core precludes protein entries and unbound proteins are filtered out by dialysis, the detected membrane proteins are most likely anchored in the bilayered lipid membranes that surround the nanoparticles.” Encouragingly, the RBC membrane-coated nanoparticles were in addition found to be as stable in serum as PEG-functionalized lipid-polymer hybrid nanoparticles.
To study the systemic circulation time of the RBC membrane-coated nanoparticles in comparison with those of PEG-functionalized lipid-polymer hybrid nanoparticles, and bare PLGA nanoparticles, each type of particle was loaded with hydrophobic DiD fluorescent dye, which is used as a marker for nanoparticle circulation studies. The dye-loaded constructs were then injected into the same strain of mice from which the RBC membranes were originally derived.
RBC membrane-coated nanoparticles demonstrated much better blood retention in comparison with PEG-functionalized nanoparticles, the researchers report. At 24 and 48 hours, the RBC membrane-coated nanoparticles exhibited 29% and 16% overall retention, respectively, compared with 11% and 2% retention exhibited by the PEG-coated nanoparticles at the same time points. Bare PLGA nanoparticles showed a negligible signal by two minutes, which was expected because of their rapid aggregations in serum.
Estimates further suggested that the elimination half life of the RBC membrane-coated nanoparticles was also much better than that of the PEG-coated constructs. Based on the two-compartment model, the elimination half-life for the RBC membrane-coated nanoparticles was calculated as 39.6 hours, compared with 15.8 hours for the PEG-coated nanoparticles. If a nonlinear elimination model was used, in which the nanoparticle clearance rate gradually slows down over time, the first apparent half-life of the RBC membrane-coated nanoparticles would be about 9.6 hours, and that of PEG-coated nanoparticles about 6.5 hours.
The researchers finally went on to study the in vivo tissue distribution of DiD-loaded RBC membrane-coated nanoparticles at 24-, 48-, and 72-hour time points following injection. After accounting for tissue mass, it was found that the nanoparticles were primarily distributed in the blood and liver. The fluorescence signals from the blood correlate well with the data from the circulation half-life study, and demonstrated 21%, 15%, and 11% of nanoparticle retention at 24-, 48-, and 72-hour marks, respectively. As the blood fluorescence decreased, a corresponding increase in signal was observed in the liver, suggesting that the blood-borne nanoparticles were eventually taken up by the liver, the authors note.
The erythrocyte membrane-coated nanoparticles are structurally analogous to lipid-polymer hybrid nanoparticles, which are being developed as a multifunctional drug delivery platform combining the desirable characteristics of both liposomes and polymeric constructs, the researchers note. However, they expect the RBC membrane-coated nanoparticles will display even better drug release kinetics because the RBC membrane provides a more dense and bilayered lipid barrier against drug diffusion.
The authors admit that translating their mouse research to broad use in human drug delivery will present challenges, not least in because the presence of blood type-specific erythrocyte antigens means that human RBC membrane-coated nanoparticles would need to be crossmatched to patients’ blood, in a similar way to crossmatching prior to blood transfusion. “Alternatively, this biomimetic delivery platform could be an elegant method for personalized medicine whereby the drug delivery nanocarrier is tailored to individual patients with little risk of immunogenicity by using their own RBC membranes as the particle coatings,” the authors suggest.
The next step will be to develop an approach for the large-scale manufacture of RBC membrane-coated nanoparticles suitable for clinical use, the team notes. This will be funded through the National Science Foundation. Research will in addition focus on adding cancer-targeting molecules to the membrane, and loading the nanoparticle core with multiple drugs.