Huining He

Enhancing insulin oral absorption by using mucoadhesive nanoparticles loaded with LMWP-linked insulin conjugates.

Although significant progress has been achieved, effective oral delivery of protein drugs such as insulin by nanoparticle-based carrier systems still faces certain formidable challenges. Considerable amount of protein drug is released from the nanoparticles (NPs) in the gastrointestinal (GI) tract. Because of their low permeability through the intestinal mucosa, the released protein would be soon degraded by the large amount of proteases in the GI tract. Herein, we report an oral insulin delivery system that can overcome the above-mentioned problems by mucoadhesive NPs (MNPs) loaded with cell penetrating peptide-linked insulin conjugates. On one hand, after conjugation with low molecular weight protamine (LMWP), a cell penetrating peptide (CPP), insulin showed greatly improved permeability through intestinal mucus layer and epithelia. On the other hand, the mucoadhesive N-trimethyl chitosan chloride-coated PLGA nanoparticles (MNPs) that were loaded with conjugates enhanced the retention in the intestinal mucus layer. By adopting this delivery strategy, the LMWP-insulin conjugates released from the MNPs could be deprived from enzymatic degradation, due to the short distance in reaching the epithelia and the high permeation of the conjugates through epithelia. The oral delivery system of insulin designed by us showed a long-lasting hypoglycemia effect with a faster onset in diabetic rats. The pharmacological availability of orally delivered conjugates-loaded MNPs was 17.98±5.61% relative to subcutaneously injected insulin solution, with a 2-fold higher improvement over that by MNPs loaded with native insulin. Our results suggested that conjugation with CPP followed by encapsulation in MNPs provides an effective strategy for oral delivery of macromolecular therapeutics.

Long-Circulating Heparin-Functionalized Magnetic Nanoparticles for Potential Application as a Protein Drug Delivery Platform

Starch-coated, PEGylated, and heparin-functionalized iron oxide magnetic nanoparticles (DNPH) were successfully synthesized and characterized in detail. The PEGylation (20 kDa) process resulted in an average coating of 430 PEG molecules per nanoparticle. After that, heparin conjugation was carried out to attain the final DNPH platform with 35.4 μg of heparin/mg of Fe. Commercially acquired heparin-coated magnetic nanoparticles were also PEGylated (HP) and characterized for comparison. Protamine was selected as a model protein to demonstrate the strong binding affinity and high loading content of DNPH for therapeutically relevant cationic proteins. DNPH showed a maximum loading of 22.9 μg of protamine/mg of Fe. In the pharmacokinetic study, DNPH displayed a long-circulating half-life of 9.37 h, 37.5-fold longer than that (0.15 h) of HP. This improved plasma stability enabled extended exposure of DNPH to the tumor lesions, as was visually confirmed in a flank 9L-glioma mouse model using magnetic resonance imaging (MRI). Quantitative analysis of the Fe content in excised tumor lesions further demonstrated the superior tumor targeting ability of DNPH, with up to 31.36 μg of Fe/g of tissue (13.07% injected dose (I.D.)/g of tissue) and 7.5-fold improvement over that (4.27 μg of Fe/g of tissue; 1.78% I.D./g of tissue) of HP. Overall, this study shed light on the potential of DNPH to be used as a protein drug delivery platform.

Enzyme-triggered, cell penetrating peptide-mediated delivery of anti-tumor agents

Conventional chemotherapy has little or no specificity for cancer cells, normally resulting in low drug accumulation at the tumor region (inefficacy) and drug-induced severe side effects (toxicity). Nowadays, new strategies have been developed to improve both the targeting ability and cellular drug uptake using active targeting ligands and drug internalization agents, which could recognize and interact with specific receptors overexpressed on tumor cells and then trigger a drug internalization process by transporting the cargos into cells. Among those strategies, enzyme-triggered cell penetrating peptide (CPP)-mediated systems seem to be a feasible approach. The expression level of specific enzymes like proteases, esterases or glycosidases is often higher in tumor cells than in normal tissues, and such concentration gradients can be exploited as a tool for targeted cancer therapy. CPPs are known to be effective in promoting membrane transportation of the drug cargos, rendering a deeper tumor permeation that could further enhance the therapeutic efficacy of the delivered drug. An enzyme-triggered, CPP-mediated system would combine these advantages to yield a system with the enhanced tumor targeting ability and internalization efficiency and so far many systems have been successfully exploited and applied to cancer therapy. In this review, typical enzymes applied in cancer theranostic systems were firstly reviewed, followed by analyzing pros and cons of cell penetrating peptides. Most importantly, different types of applications of enzyme-triggered CPP-mediated systems in tumor imaging were illustrated. Finally, the drug loaded applications, i.e. enzyme-triggered CPP-mediated systems in drug delivery were reviewed.

In vivo delivery of cell-permeable antisense hypoxia-inducible factor 1α oligonucleotide to adipose tissue reduces adiposity in obese mice

Ongoing research has gradually recognized and understood the importance of adipose tissue (AT) angiogenesis as a key modulating factor of adipogenesis in the development of obesity. Previously, we carried out the first in vitro demonstration of the down-regulation of hypoxic angiogenesis during adipogenesis using cell-permeable chemical conjugates composed of antisense hypoxia-inducible factor 1α (HIF1α) oligonucleotide (ASO) and low-molecular weight protamine (LMWP). To further confirm the in vivo feasibility, we administered ASO-LMWP conjugates (AL) to diet-induced obese (DIO) mice by intraperitoneal injection (IP). Results showed that the AL conjugates significantly reduced the body weight, total fat tissue weight, and plasma lipid concentrations in the mice. Moreover, the AL conjugates not only decreased liver weight and hepatic triglyceride concentration but also significantly attenuated subcutaneous adipocyte cell size, which was conversely increased in the AL-untreated high-fat diet (HFD) group. Interestingly, more blood vessels were observed in the HFD group than in the lean group, indicating that blood vessel development could induce growth of the fat mass. This pattern was reversed in the AL-treated groups, which displayed a decrease in blood vessel density compared to the AL-untreated HFD group. This study presents the first in vivo evidence, in an obese mouse model, of the feasibility of achieving a biological treatment modality for obesity by blocking the angiogenic transcriptional factor HIF1α, thereby limiting angiogenesis, via the use of an adipose tissue-permeable ASO-LMWP.

A Prodrug-type, MMP-2-targeting Nanoprobe for Tumor Detection and Imaging

Tumor-associated proteases (TAPs) have been intensively studied because of their critical roles in cancer development. As a case in point, expression of matrix metalloproteases (MMP) is significantly up-regulated in tumorigenesis, invasion, and metastasis among a majority of cancers. Here we present a prodrug-type, MMP-2-responsive nanoprobe system with high efficiency and low toxicity for detecting MMP-2-overexpressed tumors. The nanoprobe system is featured by its self-assembled fabrication and FRET effect. This prodrug-type nanoprobe is selectively activated by MMP-2, and thus useful for detection of the MMP-2-overexpressed cells and tumors. The nanoprobe system works successfully in various animal tumor models, including human fibrosarcoma and subcutaneous glioma xenograft. Furthermore, in order to overcome the blood brain barrier (BBB) and achieve brain tumor targeting, a transferrin-receptor targeting peptide (T7 peptide) is strategically incorporated into the nanoprobe. The T7-functionalized nanoprobe is capable of detecting the orthotopic brain tumor, with clear, real-time in vivo imaging. This method is promising for in vivo detection of brain tumor, and real-time monitor of a TAP (i.e., MMP-2).

Specific down regulation of 3T3-L1 adipocyte differentiation by cell-permeable antisense HIF1α-oligonucleotide

Hypoxia is a strong modulator of angiogenesis, accelerating adipose tissue expansion, suggesting that hypoxia inducible factor 1alpha (HIF1alpha) can be a novel target for anti-obesity. We conjugated antisense-HIF1alpha-oligonucleotide (ASO) with low molecular weight protamine (LMWP), a cell-penetrating peptide, to enhance its ability to block hypoxic-angiogenesis, thereby eliciting an anti-obesity effect. Nano-sized ASO-LMWP (AS-L) conjugates enhanced cellular uptake of ASO without yielding a cytotoxic effect and protected the ASO against enzymatic attack and chemical reduction. AS-L showed enhanced intra-cellular localization compared to naked ASO and the complex of ASO with lipofectamine during hypoxic-differentiation. Consequently AS-L induced significant down-regulation of leptin and VEGF gene expressions, thereby reducing fat accumulation in the cell. This proof-of-concept study shows that AS-L produces an inhibitory effect on adipogenesis and angiogenesis during differentiation, indicating LMWP mediated ASO delivery can potentially be a safe and promising treatment for obesity.

CPP-Assisted Intracellular Drug Delivery, What Is Next?

For the past 20 years, we have witnessed an unprecedented and, indeed, rather miraculous event of how cell-penetrating peptides (CPPs), the naturally originated penetrating enhancers, help overcome the membrane barrier that has hindered the access of bio-macromolecular compounds such as genes and proteins into cells, thereby denying their clinical potential to become potent anti-cancer drugs. By taking the advantage of the unique cell-translocation property of these short peptides, various payloads of proteins, nucleic acids, or even nanoparticle-based carriers were delivered into all cell types with unparalleled efficiency. However, non-specific CPP-mediated cell penetration into normal tissues can lead to widespread organ distribution of the payloads, thereby reducing the therapeutic efficacy of the drug and at the same time increasing the drug-induced toxic effects. In view of these challenges, we present herein a review of the new designs of CPP-linked vehicles and strategies to achieve highly effective yet less toxic chemotherapy in combating tumor oncology.

Recombinant TAT–gelonin fusion toxin: Synthesis and characterization of heparin/protamine‐regulated cell transduction

Protein toxins, such as gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TAT-Gel displayed up to 177-fold lower IC₅₀ (avg. 54.3 nM) than rGel (avg. IC₅₀ : 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TAT-Gel in achieving a highly effective yet safe drug therapy for the treatment of tumors.

Nose-to-brain delivery of macromolecules mediated by cell-penetrating peptides.

Brain delivery of macromolecular therapeutics (e.g., proteins) remains an unsolved problem because of the formidable blood–brain barrier (BBB). Although a direct pathway of nose-to-brain transfer provides an answer to circumventing the BBB and has already been intensively investigated for brain delivery of small drugs, new challenges arise for intranasal delivery of proteins because of their larger size and hydrophilicity. In order to overcome the barriers and take advantage of available pathways (e.g., epithelial tight junctions, uptake by olfactory neurons, transport into brain tissues, and intra-brain diffusion), a low molecular weight protamine (LMWP) cell-penetrating peptide was utilized to facilitate nose-to-brain transport. Cell-penetrating peptides (CPP) have been widely used to mediate macromolecular delivery through many kinds of biobarriers. Our results show that conjugates of LMWP–proteins are able to effectively penetrate into the brain after intranasal administration. The CPP-based intranasal method highlights a promising solution for protein therapy of brain diseases.

A novel strategy to achieve effective drug delivery: exploit cells as carrier combined with nanoparticles

Abstract Cell-mediated drug delivery systems employ specific cells as drug vehicles to deliver drugs to targeted sites. Therapeutics or imaging agents are loaded into these cells and then released in diseased sites. These specific cells mainly include red blood cells, leukocytes, stem cells and so on. The cell acts as a Trojan horse to transfer the drug from circulating blood to the diseased tissue. In such a system, these cells keep their original properties, which allow them to mimic the migration behavior of specific cells to carry drug to the targeted site after in vivo administration. This strategy elegantly combines the advantages of both carriers, i.e. the adjustability of nanoparticles (NPs) and the natural functions of active cells, which therefore provides a new perspective to challenge current obstacles in drug delivery. This review will describe a fundamental understanding of these cell-based drug delivery systems, and discuss the great potential of combinational application of cell carrier and NPs.