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Self-Assembled, Dilution-Responsive Hydrogels for Enhanced Thermal Stability of Insulin

Self-Assembled, Dilution-Responsive Hydrogels for Enhanced Thermal Stability of Insulin Biopharmaceuticals

Biotherapeutics currently dominate the landscape of new drugs because of their exceptional potency and selectivity. Yet, the intricate molecular structures that give rise to these beneficial qualities also render them unstable in formulation. Hydrogels have shown potential as stabilizing excipients for biotherapeutic drugs, providing protection against harsh thermal conditions experienced during distribution and storage. In this work, they report the utilization of a cellulose-based supramolecular hydrogel formed from polymer–nanoparticle (PNP) interactions to encapsulate and stabilize insulin, an important biotherapeutic used widely to treat diabetes. Encapsulation of insulin in these hydrogels prevents insulin aggregation and maintains insulin bioactivity through stressed aging conditions of elevated temperature and continuous agitation for over 28 days. Further, insulin can be easily recovered by dilution of these hydrogels for administration at the point of care. This supramolecular hydrogel system shows promise as a stabilizing excipient to reduce the cold chain dependence of insulin and other biotherapeutics.


Biotherapeutics have grown to be a dominant portion of the pharmaceutical market over the past two decades. In 2018, eight of the top ten grossing drug products globally were biotherapeutics. While the complex and intricate structures of these macromolecular drugs result in pharmaceutical products with exceptional potency and selectivity, it also makes them inherently susceptible to loss of activity due to thermal denaturation and aggregation. Exposure to high, or even ambient, temperatures during transportation, storage, and use can cause partial or complete loss of bioactivity. To prevent loss of activity, most biotherapeutics require refrigerated shipping and storage throughout the supply chain, referred to as the “cold chain”, which reduces accessibility to regions of the world with limited infrastructure. Additionally, cold chain logistics are costly, with an estimated $15.7 billion spent globally by biopharmaceutical companies on cold chain logistics alone in 2019.


Improved thermal stability of biotherapeutics would enable increased global access to critical drugs and reduce storage and transportation costs.There are many approaches to improving the stability of biotherapeutic formulations, but typical strategies include modifying the drug molecule itself, altering its physical state, or formulating with excipients—often in combination—such as salts, amino acids, polymers, and surfactants. One prevalent method is to use protein engineering to design biotherapeutic analogues.While this has been successful, it carries the risk of altered drug potency or immunogenicity. Another commonly used stabilization technique is lyophilization, or freeze-drying, of the drug into a dry powder prior to shipping and long-term storage.Unfortunately, lyophilization is not ideal for many drug products because the drastic changes in temperature, pressure, and hydration state during processing can result in activity loss.


Liquid formulations of biotherapeutics incorporate stabilizing excipients; however, many currently used pharmaceutical excipients were developed for use with small molecule drugs and were not optimized to stabilize proteins. Therefore, there is a need to develop next-generation excipients specifically designed to enhance the stability of biotherapeutics, especially for parenteral formulations. The drug delivery field has made considerable advances in the formulation of biotherapeutics in soft materials to modulate drug pharmacokinetics; this body of knowledge and materials could be leveraged to advance materials for biostability as well.

Recently, hydrogels have been utilized to improve the thermal stability of biotherapeutics. Nanogels, nanoparticles, microparticles, and hydrogel films are forms of hydrogel excipients used to stabilize the protein of interest and can reduce cold chain dependence.


Hydrogels are promising candidates for biotherapeutic stabilization because they can maintain the proteins in their native aqueous environment while promoting stabilizing interactions between the hydrogel polymer backbone and protein. A poly(ethylene glycol) (PEG)-based covalent hydrogel network with a photolabile moiety has been reported to stabilize biotherapeutic cargo at elevated temperatures. Upon exposure to ultraviolet light, the hydrogel network backbone degraded to afford biotherapeutic recovery. While the system effectively stabilized the selected enzymes, the presence of degraded hydrogel material may prove to be an obstacle for clinical translation as the in vivo biocompatibility has not been assessed. Both trehalose-based hydrogels and hydrogels with trehalose side chain modifications have also shown utility as thermal stabilizing excipients for a variety of proteins, including insulin. Unfortunately, these systems have not been shown to be injectable or degradable in vivo. To use hydrogels as stabilizing excipients in parenteral biotherapeutic formulations, there is still a need to design materials that allow mild encapsulation, are injectable, are biocompatible in vivo, and easily release the drug without unintentionally altering drug pharmacokinetics.


They have developed a supramolecular hydrogel platform, based upon polymer–nanoparticle (PNP) interactions between hydrophobically modified cellulose-derived polymers and core–shell polymeric nanoparticles, as an encapsulating excipient.These supramolecular hydrogels have been used for a variety of biomedical applications and are biocompatible, injectable, and readily tunable in mechanical properties as characterized by rheology. They can readily encapsulate biotherapeutic cargo into these hydrogels under mild conditions by simply mixing the drug into the bulk material. Further, the hydrogel can be diluted to disrupt the network and yield a drug-containing solution for administration, as the various hydrogel components are Generally Recognized as Safe (GRAS-listed) by the FDA and are non-toxic. These qualities indicate that our PNP hydrogel network is a promising candidate for a translatable stabilizing excipient for biotherapeutics.


(a) Supramolecular polymer–nanoparticle (PNP) hydrogels composed of (hydroxypropyl)methyl cellulose (HPMC) with dodecyl side chain modifications as the network polymer that is physically cross-linked by bridging interactions with poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA) diblock copolymer nanoparticles. Biotherapeutics, such as insulin (shown), can be encapsulated in these hydrogels during precursor mixing. (b) Schematic indicating the desired performance of our thermally stabilizing supramolecular hydrogel excipient.


In this study, they demonstrate that PNP hydrogels can stabilize insulin against thermal aggregation and denaturation. Insulin is a clinically relevant biotherapeutic that is required by over 150 million patients living with diabetes worldwide, but suffers from thermal instability and is highly prone to aggregate into insoluble, inactive and immunogenic amyloid fibrils when it is exposed to increased temperatures or agitation. Stabilized insulin capable of maintaining drug integrity in environmental conditions could increase access to this drug in regions of the world such as Africa and the Western Pacific where diabetes prevalence is increasing but reliable cold chain storage and transportation is challenging. They investigate the thermal stability of insulin encapsulated in these hydrogels with stressed aging experiments under relevant simulated environmental conditions to demonstrate the possibility for cold chain independence. They show that these supramolecular hydrogels are promising for use as thermal stabilization agents in insulin formulations, and potentially other parenteral biotherapeutic formulations as well.



Rheological characterization of 1 wt % polymer and 2 wt % polymer PNP hydrogel formulations with varying wt % NPs. Hydrogel formulations are denoted as P:NP, where P = wt % polymer and NP = wt % nanoparticles. Neat polymer solutions are denoted as 1:0 and 2:0 formulations. (a) Frequency-dependent oscillatory shear measurements indicate the hydrogel storage (G′) and loss (G″) moduli increase with increasing wt % NPs. (b) Steady-shear measurements of all hydrogel formulations exhibit significant shear-thinning behavior that collapses to the shear viscosity values of the neat polymer solutions.



Supramolecular hydrogels can be diluted to disrupt the gel network and release drug cargo. (a, b) For the stiffest gel formulation (2:10), 4× dilution with PBS buffer allows the material to flow. Bar chart data at 1 s–1 shear rate. (c) Inverted vial test indicating an approximate gel-to-sol transition upon 4× dilution of the 2:10 gel formulation. (d, e) For the significantly less stiff 2:2 gel formulation, a 2× dilution with PBS buffer allows the material to flow. (f) Inverted vial test indicating an approximate gel-to-sol transition upon 2× dilution of the 2:2 gel formulation.


  1. Self-Assembled, Dilution-Responsive Hydrogels for Enhanced Thermal Stability of Insulin Biopharmaceuticals Catherine M. Meis, Erika E. Salzman, Caitlin L. Maikawa, Anton A. A. Smith, Joseph L. Mann, Abigail K. Grosskopf, and Eric A. Appel ACS Biomaterials Science & Engineering 2021 7 (9), 4221-4229 DOI: 10.1021/acsbiomaterials.0c01306