Novel System for the Stabilization and Delivery of Proteins to the Insect Hemocoel through Conjugation with Aliphatic Polyethylene Glycol

Despite the numerous insecticidal proteins identified, there are few commercially-successful protein insecticides. Oral delivery of proteins is difficult due to their degradation by digestive endo- and exo-peptidases and their limited movement across the gut epithelium. PEGylation, the process of covalently attaching polyethylene glycol (PEG) polymer chains to another molecule, has been used in the pharmaceutical industry for decades to deliver proteins across the digestive system of humans. In this study, two PEGylated insulins and a PEGylated insecticidal decapeptide, Trypsin Modulating Oostatic Factor (TMOF), were created to determine if PEGylation could reduce the rate of degradation and enhance the accumulation of the parent compounds from the diet or cuticle in the insect hemocoel. The chemistry for the synthesis of monodispersed aliphatic TMOF-K-PEG₇P, polydispersed aliphatic PEG350-insulin and monodispersed aliphatic PEG333-insulin are described herein. The PEGylation of insulin yields a 6.7 and 7.3 fold increase in appearance of insulin species in the hemolymph of Heliothis virescens larvae after feeding for the PEG350 and PEG333 chemistries, respectively. When insulin is topically applied to the dorsum of H. virescens, no insulin is found in the hemolymph. However, after topical application of the PEGylated insulins, insulin species were detected in the hemolymph. After injections of insulin into the hemocoel of 4th stadium H. virescens, insulin is completely cleared from the hemolymph in 120 minutes. In comparison, when PEG350-insulin and PEG333-insulin are injected into the hemocoel, insulin species were still present in the hemolymph 300 and 240 minutes after injection, respectively, translating to a 3.3 and 2.7 fold increases in the length of time insulin remains in the hemolymph after injection. Conjugation of TMOF to polyethylene glycol increased its insecticidal effects against several insect species. For example, the addition of lysine to TMOF reduced its per os activity relative to the parent TMOF, but conjugation of TMOF-K with methyl(ethyleneglycol)₇-O-propionyl increased it toxicity 5.8 and 10.1 fold above that of TMOF and TMOF-K for Aedes aegypti. However, unlike the PEGylated insulin species, no TMOF, TMOF-K, or PEGylated TMOF-K was detected in the hemolymph after topical application to the cuticle.