All Publications

  • An ultrafast insulin formulation enabled by high-throughput screening of engineered polymeric excipients. Science translational medicine Mann, J. L., Maikawa, C. L., Smith, A. A., Grosskopf, A. K., Baker, S. W., Roth, G. A., Meis, C. M., Gale, E. C., Liong, C. S., Correa, S., Chan, D., Stapleton, L. M., Yu, A. C., Muir, B., Howard, S., Postma, A., Appel, E. A. 2020; 12 (550)


    Insulin has been used to treat diabetes for almost 100 years; yet, current rapid-acting insulin formulations do not have sufficiently fast pharmacokinetics to maintain tight glycemic control at mealtimes. Dissociation of the insulin hexamer, the primary association state of insulin in rapid-acting formulations, is the rate-limiting step that leads to delayed onset and extended duration of action. A formulation of insulin monomers would more closely mimic endogenous postprandial insulin secretion, but monomeric insulin is unstable in solution using present formulation strategies and rapidly aggregates into amyloid fibrils. Here, we implement high-throughput-controlled radical polymerization techniques to generate a large library of acrylamide carrier/dopant copolymer (AC/DC) excipients designed to reduce insulin aggregation. Our top-performing AC/DC excipient candidate enabled the development of an ultrafast-absorbing insulin lispro (UFAL) formulation, which remains stable under stressed aging conditions for 25 ± 1 hours compared to 5 ± 2 hours for commercial fast-acting insulin lispro formulations (Humalog). In a porcine model of insulin-deficient diabetes, UFAL exhibited peak action at 9 ± 4 min, whereas commercial Humalog exhibited peak action at 25 ± 10 min. These ultrafast kinetics make UFAL a promising candidate for improving glucose control and reducing burden for patients with diabetes.

    View details for DOI 10.1126/scitranslmed.aba6676

    View details for PubMedID 32611683

  • A co-formulation of supramolecularly stabilized insulin and pramlintide enhances mealtime glucagon suppression in diabetic pigs. Nature biomedical engineering Maikawa, C. L., Smith, A. A., Zou, L. n., Roth, G. A., Gale, E. C., Stapleton, L. M., Baker, S. W., Mann, J. L., Yu, A. C., Correa, S. n., Grosskopf, A. K., Liong, C. S., Meis, C. M., Chan, D. n., Troxell, M. n., Maahs, D. M., Buckingham, B. A., Webber, M. J., Appel, E. A. 2020


    Treatment of patients with diabetes with insulin and pramlintide (an amylin analogue) is more effective than treatment with insulin only. However, because mixtures of insulin and pramlintide are unstable and have to be injected separately, amylin analogues are only used by 1.5% of people with diabetes needing rapid-acting insulin. Here, we show that the supramolecular modification of insulin and pramlintide with cucurbit[7]uril-conjugated polyethylene glycol improves the pharmacokinetics of the dual-hormone therapy and enhances postprandial glucagon suppression in diabetic pigs. The co-formulation is stable for over 100 h at 37 °C under continuous agitation, whereas commercial formulations of insulin analogues aggregate after 10 h under similar conditions. In diabetic rats, the administration of the stabilized co-formulation increased the area-of-overlap ratio of the pharmacokinetic curves of pramlintide and insulin from 0.4 ± 0.2 to 0.7 ± 0.1 (mean ± s.d.) for the separate administration of the hormones. The co-administration of supramolecularly stabilized insulin and pramlintide better mimics the endogenous kinetics of co-secreted insulin and amylin, and holds promise as a dual-hormone replacement therapy.

    View details for DOI 10.1038/s41551-020-0555-4

    View details for PubMedID 32393892

  • Engineering Insulin Cold Chain Resilience to Improve Global Access. Biomacromolecules Maikawa, C. L., Mann, J. L., Kannan, A., Meis, C. M., Grosskopf, A. K., Ou, B. S., Autzen, A. A., Fuller, G. G., Maahs, D. M., Appel, E. A. 2021


    There are 150 million people with diabetes worldwide who require insulin replacement therapy, and the prevalence of diabetes is rising the fastest in middle- and low-income countries. The current formulations require costly refrigerated transport and storage to prevent loss of insulin integrity. This study shows the development of simple "drop-in" amphiphilic copolymer excipients to maintain formulation integrity, bioactivity, pharmacokinetics, and pharmacodynamics for over 6 months when subjected to severe stressed aging conditions that cause current commercial formulation to fail in under 2 weeks. Further, when these copolymers are added to Humulin R (Eli Lilly) in original commercial packaging, they prevent insulin aggregation for up to 4 days at 50 °C compared to less than 1 day for Humulin R alone. These copolymers demonstrate promise as simple formulation additives to increase the cold chain resilience of commercial insulin formulations, thereby expanding global access to these critical drugs for treatment of diabetes.

    View details for DOI 10.1021/acs.biomac.1c00474

    View details for PubMedID 34213889

  • Full closed loop open-source algorithm performance comparison in pigs with diabetes. Clinical and translational medicine Lal, R. A., Maikawa, C. L., Lewis, D., Baker, S. W., Smith, A. A., Roth, G. A., Gale, E. C., Stapleton, L. M., Mann, J. L., Yu, A. C., Correa, S., Grosskopf, A. K., Liong, C. S., Meis, C. M., Chan, D., Garner, J. P., Maahs, D. M., Buckingham, B. A., Appel, E. A. 2021; 11 (4): e387


    Understanding how automated insulin delivery (AID) algorithm features impact glucose control under full closed loop delivery represents a critical step toward reducing patient burden by eliminating the need for carbohydrate entries at mealtimes. Here, we use a pig model of diabetes to compare AndroidAPS and Loop open-source AID systems without meal announcements. Overall time-in-range (70-180mg/dl) for AndroidAPS was 58% ± 5%, while time-in-range for Loop was 35% ± 5%. The effect of the algorithms on time-in-range differed between meals and overnight. During the overnight monitoring period, pigs had an average time-in-range of 90% ± 7% when on AndroidAPS compared to 22% ± 8% on Loop. Time-in-hypoglycemia also differed significantly during the lunch meal, whereby pigs running AndroidAPS spent an average of 1.4% (+0.4/-0.8)% in hypoglycemia compared to 10% (+3/-6)% for those using Loop. As algorithm design for closed loop systems continues to develop, the strategies employed in the OpenAPS algorithm (known as oref1) as implemented in AndroidAPS for unannounced meals may result in a better overall control for full closed loop systems.

    View details for DOI 10.1002/ctm2.387

    View details for PubMedID 33931977

  • Controlling properties of thermogels by tuning critical solution behaviour of ternary copolymers dagger POLYMER CHEMISTRY Smith, A. A., Maikawa, C. L., Lopez Hernandez, H., Appel, E. A. 2021

    View details for DOI 10.1039/d0py01696a

    View details for Web of Science ID 000633040700001

  • Enhanced Humoral Immune Response by High Density TLR Agonist Presentation on Hyperbranched Polymers ADVANCED THERAPEUTICS Liong, C. S., Smith, A. A., Mann, J. L., Roth, G. A., Gale, E. C., Maikawa, C. L., Ou, B. S., Appel, E. A. 2021
  • Seasonal Impact of Phosphate-Based Fire Retardants on Soil Chemistry Following the Prophylactic Treatment of Vegetation. Environmental science & technology Yu, A. C., Reinhart, M., Hunter, R., Lu, K., Maikawa, C. L., Rajakaruna, N., Acosta, J. D., Stubler, C., Appel, C., Appel, E. A. 2021


    A preventative treatment of fire retardants at high-risk locales can potentially stop a majority of wildfires. For example, over 80% of wildfire ignitions in California occur at high-risk locales such as adjacent to roadsides and utility infrastructure. Recently a new class of ammonium polyphosphate retardants was developed with enhanced adherence and retention on vegetation to enable prophylactic treatments of these high-risk locals to provide season-long prevention of ignitions. Here, we compare three different ammonium (poly)phosphate-based wildland retardant formulations and evaluate their resistance to weathering and analyze their seasonal impact on soil chemistry following application onto grass. Soil samples from all three treatments demonstrated no changes in soil pH and total soil carbon and nitrogen amounts. Total soil phosphorus amounts increased by 2-3* following early precipitation, always remaining within typical topsoil amounts, and returned to the same level as control soil before spring. Available indices of ammonium, nitrate, and phosphate levels for all groups were elevated compared to the untreated control samples, again remaining within typical topsoil ranges across all time points and rainfall amounts evaluated. Microbial activity was decreased, potentially because the addition of available nutrients from retardant application reduced the need for organic decomposition. These results demonstrate that the application of ammonium (poly)phosphate-based retardants does not alter soil chemistry beyond typical topsoil compositions and are thus suitable for use in prophylactic wildfire prevention strategies.

    View details for DOI 10.1021/acs.est.0c05472

    View details for PubMedID 33529000

  • Engineering biopharmaceutical formulations to improve diabetes management. Science translational medicine Maikawa, C. L., d'Aquino, A. I., Lal, R. A., Buckingham, B. A., Appel, E. A. 2021; 13 (578)


    Insulin was first isolated almost a century ago, yet commercial formulations of insulin and its analogs for hormone replacement therapy still fall short of appropriately mimicking endogenous glycemic control. Moreover, the controlled delivery of complementary hormones (such as amylin or glucagon) is complicated by instability of the pharmacologic agents and complexity of maintaining multiple infusions. In this review, we highlight the advantages and limitations of recent advances in drug formulation that improve protein stability and pharmacokinetics, prolong drug delivery, or enable alternative dosage forms for the management of diabetes. With controlled delivery, these formulations could improve closed-loop glycemic control.

    View details for DOI 10.1126/scitranslmed.abd6726

    View details for PubMedID 33504649

  • A fluorescence sandwich immunoassay for the real-time continuous detection of glucose and insulin in live animals. Nature biomedical engineering Poudineh, M., Maikawa, C. L., Ma, E. Y., Pan, J., Mamerow, D., Hang, Y., Baker, S. W., Beirami, A., Yoshikawa, A., Eisenstein, M., Kim, S., Vuckovic, J., Appel, E. A., Soh, H. T. 2020


    Biosensors that continuously measure circulating biomolecules in real time could provide insights into the health status of patients and their response to therapeutics. But biosensors for the continuous real-time monitoring of analytes in vivo have only reached nanomolar sensitivity and can measure only a handful of molecules, such as glucose and blood oxygen. Here we show that multiple analytes can be continuously and simultaneously measured with picomolar sensitivity and sub-second resolution via the integration of aptamers and antibodies into a bead-based fluorescence sandwich immunoassay implemented in a custom microfluidic chip. After an incubation time of 30s, bead fluorescence is measured using a high-speed camera under spatially multiplexed two-colour laser illumination. We used the assay for continuous quantification of glucose and insulin concentrations in the blood of live diabetic rats to resolve inter-animal differences in the pharmacokinetic response to insulin as well as discriminate pharmacokinetic profiles from different insulin formulations. The assay can be readily modified to continuously and simultaneously measure other blood analytes in vivo.

    View details for DOI 10.1038/s41551-020-00661-1

    View details for PubMedID 33349659

  • Site-selective modification of proteins using cucurbit[7]uril as supramolecular protection for N-terminal aromatic amino acids. Organic & biomolecular chemistry Smith, A. A., Maikawa, C. L., Roth, G. A., Appel, E. A. 2020


    Cucurbit[7,8]urils are known to form inclusion complexes with aromatic amino acids, hosting the hydrohobic side chains within the cavity and adjacent cations within the portal of the macrocyclic host. Here we show that cucurbit[7]uril binding with N-terminal phenylalanine significantly reduces the nucleophilicity of the amine, likely due to an increase in stability of the ammonium ion, rendering it unreactive at neutral pH. Using insulin as a model protein, we show that this supramolecular protection strategy can drive selectivity of N-terminal amine conjugation away from the preferred B chain N-terminal phenylalanine towards the A chain N-terminal glycine. Cucurbit[7]uril can therefore be used as a supramolecular protecting group for site-selective protein modification.

    View details for DOI 10.1039/d0ob01004a

    View details for PubMedID 32459261

  • Stable Monomeric Insulin Formulations Enabled by Supramolecular PEGylation of Insulin Analogues. Advanced therapeutics Maikawa, C. L., Smith, A. A., Zou, L. n., Meis, C. M., Mann, J. L., Webber, M. J., Appel, E. A. 2020; 3 (1)


    Current "fast-acting" insulin analogues contain amino acid modifications meant to inhibit dimer formation and shift the equilibrium of association states toward the monomeric state. However, the insulin monomer is highly unstable and current formulation techniques require insulin to primarily exist as hexamers to prevent aggregation into inactive and immunogenic amyloids. Insulin formulation excipients have thus been traditionally selected to promote insulin association into the hexameric form to enhance formulation stability. This study exploits a novel excipient for the supramolecular PEGylation of insulin analogues, including aspart and lispro, to enhance the stability and maximize the prevalence of insulin monomers in formulation. Using multiple techniques, it is demonstrated that judicious choice of formulation excipients (tonicity agents and parenteral preservatives) enables insulin analogue formulations with 70-80% monomer and supramolecular PEGylation imbued stability under stressed aging for over 100 h without altering the insulin association state. Comparatively, commercial "fast-acting" formulations contain less than 1% monomer and remain stable for only 10 h under the same stressed aging conditions. This simple and effective formulation approach shows promise for next-generation ultrafast insulin formulations with a short duration of action that can reduce the risk of post-prandial hypoglycemia in the treatment of diabetes.

    View details for DOI 10.1002/adtp.201900094

    View details for PubMedID 32190729

    View details for PubMedCentralID PMC7079736

  • Nanoparticles Presenting Potent TLR7/8 Agonists Enhance Anti-PD-L1 Immunotherapy in Cancer Treatment. Biomacromolecules Smith, A. A., Gale, E. C., Roth, G. A., Maikawa, C. L., Correa, S. n., Yu, A. C., Appel, E. A. 2020


    Cancer immunotherapy can be augmented with toll-like receptor agonist (TLRa) adjuvants, which interact with immune cells to elicit potent immune activation. Despite their potential, use of many TLRa compounds has been limited clinically due to their extreme potency and lack of pharmacokinetic control, causing systemic toxicity from unregulated systemic cytokine release. Herein, we overcome these shortcomings by generating poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) nanoparticles (NPs) presenting potent TLR7/8a moieties on their surface. The NP platform allows precise control of TLR7/8a valency and resulting surface presentation through self-assembly using nanoprecipitation. We hypothesize that the pharmacokinetic profile of the NPs minimizes systemic toxicity, localizing TLR7/8a presentation to the tumor bed and tumor-draining lymph nodes. In conjunction with antiprogrammed death-ligand 1 (anti-PD-L1) checkpoint blockade, peritumoral injection of TLR7/8a NPs slows tumor growth, extends survival, and decreases systemic toxicity in comparison to the free TLR7/8a in a murine colon adenocarcinoma model. These NPs constitute a modular platform for controlling pharmacokinetics of immunostimulatory molecules, resulting in increased potency and decreased toxicity.

    View details for DOI 10.1021/acs.biomac.0c00812

    View details for PubMedID 32816460

  • Injectable Hydrogels for Sustained Codelivery of Subunit Vaccines Enhance Humoral Immunity. ACS central science Roth, G. A., Gale, E. C., Alcántara-Hernández, M. n., Luo, W. n., Axpe, E. n., Verma, R. n., Yin, Q. n., Yu, A. C., Lopez Hernandez, H. n., Maikawa, C. L., Smith, A. A., Davis, M. M., Pulendran, B. n., Idoyaga, J. n., Appel, E. A. 2020; 6 (10): 1800–1812


    Vaccines aim to elicit a robust, yet targeted, immune response. Failure of a vaccine to elicit such a response arises in part from inappropriate temporal control over antigen and adjuvant presentation to the immune system. In this work, we sought to exploit the immune system's natural response to extended pathogen exposure during infection by designing an easily administered slow-delivery vaccine platform. We utilized an injectable and self-healing polymer-nanoparticle (PNP) hydrogel platform to prolong the codelivery of vaccine components to the immune system. We demonstrated that these hydrogels exhibit unique delivery characteristics, whereby physicochemically distinct compounds (such as antigen and adjuvant) could be codelivered over the course of weeks. When administered in mice, hydrogel-based sustained vaccine exposure enhanced the magnitude, duration, and quality of the humoral immune response compared to standard PBS bolus administration of the same model vaccine. We report that the creation of a local inflammatory niche within the hydrogel, coupled with sustained exposure of vaccine cargo, enhanced the magnitude and duration of germinal center responses in the lymph nodes. This strengthened germinal center response promoted greater antibody affinity maturation, resulting in a more than 1000-fold increase in antigen-specific antibody affinity in comparison to bolus immunization. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of subunit vaccines.

    View details for DOI 10.1021/acscentsci.0c00732

    View details for PubMedID 33145416

    View details for PubMedCentralID PMC7596866

  • Block copolymer composition drives function of self-assembled nanoparticles for delivery of small-molecule cargo JOURNAL OF POLYMER SCIENCE PART A-POLYMER CHEMISTRY Maikawa, C. L., Sevit, A., Lin, B., Wallstrom, R. J., Mann, J. L., Yu, A. C., Waymouth, R. M., Appel, E. A. 2019; 57 (12): 1322–32

    View details for DOI 10.1002/pola.29393

    View details for Web of Science ID 000469938700008

  • Comparison of Airway Responses Induced in a Mouse Model by the Gas and Particulate Fractions of Gasoline Direct Injection Engine Exhaust INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH Maikawa, C. L., Zimmerman, N., Ramos, M., Shah, M., Wallace, J. S., Pollitt, K. 2018; 15 (3)


    Diesel exhaust has been associated with asthma, but its response to other engine emissions is not clear. The increasing prevalence of vehicles with gasoline direct injection (GDI) engines motivated this study, and the objective was to evaluate pulmonary responses induced by acute exposure to GDI engine exhaust in an allergic asthma murine model. Mice were sensitized with an allergen to induce airway hyperresponsiveness or treated with saline (non-allergic group). Animals were challenged for 2-h to exhaust from a laboratory GDI engine operated at conditions equivalent to a highway cruise. Exhaust was filtered to assess responses induced by the particulate and gas fractions. Short-term exposure to particulate matter from GDI engine exhaust induced upregulation of genes related to polycyclic aromatic hydrocarbon (PAH) metabolism (Cyp1b1) and inflammation (TNFα) in the lungs of non-allergic mice. High molecular weight PAHs dominated the particulate fraction of the exhaust, and this response was therefore likely attributable to the presence of these PAHs. The particle fraction of GDI engine exhaust further contributed to enhanced methacholine responsiveness in the central and peripheral tissues in animals with airway hyperresponsiveness. As GDI engines gain prevalence in the vehicle fleet, understanding the health impacts of their emissions becomes increasingly important.

    View details for DOI 10.3390/ijerph15030429

    View details for Web of Science ID 000428509200036

    View details for PubMedID 29494515

    View details for PubMedCentralID PMC5876974