Protein microspheres: amorphously yours with SAXS

Gentle microspheres

A simple, inexpensive, and gentle process can be used to make pure protein microspheres of uniform size for therapeutic use. Microspheres of insulin for instance, shown to be amorphous by X-ray scattering (SAXS), could have advantages over other experimental delivery modes, the study's authors suggest.

Proteins represent a largely unexplored therapeutic realm with a few well-known exceptions, such as monoclonal antibodies and insulin. Not only are they expensive to make with current technology, they usually have lifetimes that are too short under physiological conditions either to allow them to reach their disease target or to persist for a sufficiently long time to have the desired therapeutic effect. However, protein drugs are worth pursuing because they demonstrate high biological activity and can be highly specific in their effects on a target tissue. Indeed, experimental work has led to increasingly tailored proteins with specific pharmacological properties that demonstrate efficacy in vitro. Transport and controlled release of the protein drugs in the body remains a significant problem.

Loading up on insulin

Loading up host species at the nano- and micro-scale in order to transport a therapeutic protein or peptide to a disease site has been a common experimental strategy that has had some success. It relies on making very uniform particles with precisely defined protein, size, composition, particle morphology, and density. These features are needed to endow the particles with high bioavailability and to control the dose rate of release once the particles reach their target site in the body. Unfortunately, such particles are difficult to control when using conventional methods for the production of particulate proteins, such as crystallization, spray drying and the incorporation of proteins into liposomes or polymer matrices. An additional problem lies in the fact that such processes commonly need toxic volatile organic solvents, high temperatures and other reaction parameters that are not conducive to the proteins remaining stable. The processes often lead to protein denaturation and a loss or reduction of biological activity as a consequence.

Now, writing in the chemistry journal Angewandte Chemie, Helmuth Moehwald of the Max Planck Institute of Colloids and Interfaces in Golm-Potsdam, and Dmitry Volodkin and Regine von Klitzing of the TU Berlin, Germany, have introduced a novel approach to the transport of proteins that sidesteps all of these issues. Their approach involves simply forming the therapeutic proteins into pure and uniform protein microspheres without a host structure or encapsulating material.

The team was hoping to avoid all the additives, solvents and reaction conditions that might denature the therapeutic protein but at the same time offer them a way to produce entirely uniform protein particles. The researchers have now successfully used porous calcium carbonate microspheres, microcores, with a defined size to template the formation of protein particles, rather as a child might use a plastic moulding to form uniform spheres of modelling clay, for instance, but obviously on a much smaller scale. The team explains that a change in pH value is key to the process. Under slightly alkaline aqueous conditions (higher pH), they found that the protein insulin is soluble. When the calcium carbonate spheres are added to an insulin solution, their pores fill with this insulin solution. Then, by simply adding acid, the team makes the insulin precipitate out of solution as insoluble protein trapped within the pores. Further acidification then dissolves the calcium carbonate spheres slowly, leaving behind spherical insulin particles that exist as a loose matrix of protein. This loos matrix subsequently shrinks into much more compact micrometre-scale spheres of uniform size and shape with a high protein density.

SAXS and the secondary

The characteristics of the insulin microspheres, as revealed by small angle X-ray scattering experiments (SAXS), suggest that the form adopted by the protein could have significant therapeutic advantages. The team explains that the collapsed protein matrix contains a significant amount of water independent of initial loading in the calcium carbonate cores, which is due to the amorphous, as opposed to crystalline, nature of the insulin in the microspheres as revealed by SAXS. Research has demonstrated that amorphous insulin may have medical advantages over the crystalline phase given that the secondary structure of the protein is unaffected by this form of precipitation. Amorphous insulin is also more stable towards chemical degradation. Such characteristics perhaps bode well for the development of this technology for the development of protein drugs beyond insulin that might be administered orally or for delivery of protein agents to the deep lung, for instance.

"The features of the protein microspheres make the microspheres valuable for protein delivery and show potential to achieve high systemic bioavailability and avoid potential complications owing to the presence of additives.

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