Enhancing Ocular Drug Delivery: The Effect of Physicochemical Properties of Nanoparticles on the Mechanism of Their Uptake by Human Cornea Epithelial Cells.

This study investigates the effect of nanoparticle size and surface chemistry on interactions of the nanoparticles with human cornea epithelial cells (HCECs). Poly(lactic-co-glycolic) acid (PLGA) nanoparticles were synthesized using the emulsion-solvent evaporation method and surface modified with mucoadhesive (alginate [ALG] and chitosan [CHS]) and mucopenetrative (polyethylene glycol [PEG]) polymers. Particles were found to be monodisperse (polydispersity index (PDI) below 0.2), spherical, and with size and zeta potential ranging from 100 to 250 nm and from -25 to +15 mV, respectively. Evaluation of cytotoxicity with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay indicated that incubating cells with nanoparticles for 24 h at concentrations up to 100 µg/mL caused only mild toxicity (70-100% cell viability). Cellular uptake studies were conducted using an in vitro model developed with a monolayer of HCECs integrated with simulated mucosal solution. Evaluation of nanoparticle uptake revealed that energy-dependent endocytosis is the primary uptake mechanism. Among the different nanoparticles studied, 100 nm PLGA NPs and PEG-PLGA-150 NPs showed the highest levels of uptake by HCECs. Additionally, uptake studies in the presence of various inhibitors suggested that macropinocytosis and caveolae-mediated endocytosis are the dominant pathways. While clathrin-mediated endocytosis was found to also be partially responsible for nanoparticle uptake, phagocytosis did not play a role within the studied ranges of size and surface chemistries. These important findings could lead to improved nanoparticle-based formulations that could improve therapies for ocular diseases.

Improving Cancer Targeting: A Study on the Effect of Dual-Ligand Density on Targeting of Cells Having Differential Expression of Target Biomarkers.

Silica nanoparticles with hyaluronic acid (HA) and folic acid (FA) were developed to study dual-ligand targeting of CD44 and folate receptors, respectively, in colon cancer. Characterization of particles with dynamic light scattering showed them to have hydrodynamic diameters of 147-271 nm with moderate polydispersity index (PDI) values. Surface modification of the particles was achieved by simultaneous reaction with HA and FA and results showed that ligand density on the surface increased with increasing concentrations in the reaction mixture. The nanoparticles showed minimal to no cytotoxicity with all formulations showing ≥ 90% cell viability at concentrations up to 100 µg/mL. Based on flow cytometry results, SW480 cell lines were positive for both receptors, the WI38 cell line was positive for CD44 receptor, and Caco2 was positive for the folate receptor. Cellular targeting studies demonstrated the potential of the targeted nanoparticles as promising candidates for delivery of therapeutic agents. The highest cellular targeting was achieved with particles synthesized using folate:surface amine (F:A) ratio of 9 for SW480 and Caco2 cells and at F:A = 0 for WI38 cells. The highest selectivity was achieved at F:A = 9 for both SW480:WI38 and SW480:Caco2 cells. Based on HA conjugation, the highest cellular targeting was achieved at H:A = 0.5-0.75 for SW480 cell, at H:A = 0.75 for WI38 cell and at H:A = 0.5 for Caco2 cells. The highest selectivity was achieved at H:A = 0 for both SW480:WI38 and SW480:Caco2 cells. These results demonstrated that the optimum ligand density on the nanoparticle for targeting is dependent on the levels of biomarker expression on the target cells. Ongoing studies will evaluate the therapeutic efficacy of these targeted nanoparticles using in vitro and in vivo cancer models.

MRI-traceable theranostic nanoparticles for targeted cancer treatment.

Current cancer therapies, including chemotherapy and radiotherapy, are imprecise, non-specific, and are often administered at high dosages - resulting in side effects that severely impact the patient's overall well-being. A variety of multifunctional, cancer-targeted nanotheranostic systems that integrate therapy, imaging, and tumor targeting functionalities in a single platform have been developed to overcome the shortcomings of traditional drugs. Among the imaging modalities used, magnetic resonance imaging (MRI) provides high resolution imaging of structures deep within the body and, in combination with other imaging modalities, provides complementary diagnostic information for more accurate identification of tumor characteristics and precise guidance of anti-cancer therapy. This review article presents a comprehensive assessment of nanotheranostic systems that combine MRI-based imaging (T1 MRI, T2 MRI, and multimodal imaging) with therapy (chemo-, thermal-, gene- and combination therapy), connecting a range of topics including hybrid treatment options (e.g. combined chemo-gene therapy), unique MRI-based imaging (e.g. combined T1-T2 imaging, triple and quadruple multimodal imaging), novel targeting strategies (e.g. dual magnetic-active targeting and nanoparticles carrying multiple ligands), and tumor microenvironment-responsive drug release (e.g. redox and pH-responsive nanomaterials). With a special focus on systems that have been tested in vivo, this review is an essential summary of the most advanced developments in this rapidly evolving field.

Development and application of high-throughput screens for the discovery of compounds that disrupt ErbB4 signaling: Candidate cancer therapeutics.

Whereas recent clinical studies report metastatic melanoma survival rates high as 30-50%, many tumors remain nonresponsive or become resistant to current therapeutic strategies. Analyses of The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) data set suggests that a significant fraction of melanomas potentially harbor gain-of-function mutations in the gene that encodes for the ErbB4 receptor tyrosine kinase. In this work, a drug discovery strategy was developed that is based on the observation that the Q43L mutant of the naturally occurring ErbB4 agonist Neuregulin-2beta (NRG2ß) functions as a partial agonist at ErbB4. NRG2ß/Q43L stimulates tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and inhibits agonist-induced ErbB4-dependent cell proliferation. Compounds that exhibit these characteristics likely function as ErbB4 partial agonists, and as such hold promise as therapies for ErbB4-dependent melanomas. Consequently, three highly sensitive and reproducible (Z' > 0.5) screening assays were developed and deployed for the identification of small-molecule ErbB4 partial agonists. Six compounds were identified that stimulate ErbB4 phosphorylation, fail to stimulate ErbB4-dependent cell proliferation, and appear to selectively inhibit ErbB4-dependent cell proliferation. Whereas further characterization is needed to evaluate the full therapeutic potential of these molecules, this drug discovery platform establishes reliable and scalable approaches for the discovery of ErbB4 inhibitors.

Evaluation of commercial soft contact lenses for ocular drug delivery: A review.

Soft contact lenses have generated growing interest in ocular drug delivery due to their potential to enhance drug bioavailability in ocular tissues. Commercially available soft contact lenses offer several advantages for ocular drug delivery as they are manufactured on a large scale, which guarantees the availability of a consistent and reproducible product, and their favorable safety profile is well-established through broad clinical use. Here we review the rationale for using commercially available soft contact lenses for ocular drug delivery; summarize the evolution of the materials used in contact lens fabrication; and explore various methods used to improve the drug release characteristics and its tissue penetration. While significant progress has been made, several issues still require further attention for the commercial launch of a viable drug-eluting contact lens product, including control of initial burst release, shelf-life stability, and drug loss during processing or storage.

Improving the Size Homogeneity of Multicore Superparamagnetic Iron Oxide Nanoparticles.

Superparamagnetic iron oxide nanoparticles (SPIONs) have been widely explored for use in many biomedical applications. Methods for synthesis of magnetic nanoparticle (MNP), however, typically yield multicore structures with broad size distribution, resulting in suboptimal and variable performance in vivo. In this study, a new method for sorting SPIONs by size, labeled diffusive magnetic fractionation (DMF), is introduced as an improvement over conventional magnetic field flow fractionation (MFFF). Unlike MFFF, which uses a constant magnetic field to capture particles, DMF utilizes a pulsed magnetic field approach that exploits size-dependent differences in the diffusivity and magnetic attractive force of SPIONs to yield more homogenous particle size distributions. To compare both methods, multicore SPIONs with a broad size distribution (polydispersity index (PdI) = 0.24 ± 0.05) were fractionated into nine different-sized SPION subpopulations, and the PdI values were compared. DMF provided significantly improved size separation compared to MFFF, with eight out of the nine fractionations having significantly lower PdI values (p value < 0.01). Additionally, the DMF method showed a high particle recovery (>95%), excellent reproducibility, and the potential for scale-up. Mathematical models were developed to enable optimization, and experimental results confirmed model predictions (R2 = 0.98).

Quantitative, real-time in vivo tracking of magnetic nanoparticles using multispectral optoacoustic tomography (MSOT) imaging.

The goal of this work was to demonstrate real-time tracking of in vivo nanoparticle concentrations utilizing multispectral optoacoustic tomography (MSOT). Combining the high contrast of optical imaging with the high resolution of ultrasound imaging, MSOT was utilized for non-invasive, real-time tomographic imaging of particles in mice and the results calibrated against analysis of tissue samples with electron paramagnetic resonance (EPR) spectroscopy. In a longitudinal study, the pharmacokinetics (pK) and biodistribution of Cyanine-7 (Cy7) conjugated superparamagnetic iron oxide nanoparticles (Cy7-SPIONs) were monitored after intravenous administration into the tail vein of healthy B6-albino mice. Concentrations of Cy7-SPIONs determined by MSOT image analysis of the liver, spleen, and kidneys showed excellent agreement with EPR data obtained on tissue samples ‒ validating MSOT's ability to quantify SPION concentrations with high spatial resolution. Both methods of analysis indicated highest accumulation of Cy7-SPIONs in the liver followed by the spleen, and negligible accumulation in the kidneys; SPION accumulation in organs with high concentrations of mononuclear phagocytic system macrophages is typical. Additionally, our study observed that particles modified with a 2 kDa polyethylene glycol (PEG) demonstrated significantly prolonged half-life in circulation compared to particles with 5 kDa PEG. The study demonstrates the potential of Cy7-SPIONs and MSOT for quantitative localization of magnetic nanoparticles in vivo, which can potentially be used to study their toxicity, quantify the efficacy of targeted drug delivery (e.g. within tumors), and their use as a multi-modal diagnostic agent to monitor disease progression.

Synthesis of silica-alginate nanoparticles and their potential application as pH-responsive drug carriers.

Composite silica-alginate nanoparticles were prepared via silica sol-gel technique using a water-in-oil microemulsion system. In our system, cyclohexane served as the bulk oil phase into which aqueous solutions of sodium alginate were dispersed as droplets that confined nanoparticle formation after addition of tetraethylorthosilicate (TEOS). Our studies showed that much of the particle growth is completed within the first 24 hours and reaction times up to 120 hours only resulted in an additional 5% increase in particle diameter. Average particle size was found to decrease with increasing water-to-surfactant molar ratio (R) and with increasing cocentration of alginate in the aqueous phase. The potential for drug loading during particle formation was demonstrated using rhodamine B as a model drug. In vitro release studies showed that particles incubated in pH 2.5 phosphate buffer released only about 7% of the drug load in 27 days, while 42% was released in pH 7.5 phosphate buffer over the same period. Analysis of the release profile suggested that rhodamine B was homogeneously distributed throughout the particle and that the drug diffusivity was 40-fold greater in pH 7.5 buffer compared to that at pH 2.5. These results suggest that silica-alginate nanoparticles could be used as a pH-responsive drug carrier for controlled drug release.

Nanoparticle-based probes to enable noninvasive imaging of proteolytic activity for cancer diagnosis.

Proteases play a key role in tumor biology, with high expression levels often correlating with poor prognosis for cancer patients - making them excellent disease markers for tumor diagnosis. Despite their significance, quantifying proteolytic activity in vivo remains a challenge. Nanoparticles, with their ability to serve as scaffolds having unique chemical, optical and magnetic properties, offer the promise of merging diagnostic medicine with material engineering. Such nanoparticles can interact preferentially with proteases enriched in tumors, providing the ability to assess disease state in a noninvasive and spatiotemporal manner. We review recent advances in the development of nanoparticles for imaging and quantification of proteolytic activity in tumor models, and prognosticate future advancements.

Exploiting Size-Dependent Drag and Magnetic Forces for Size-Specific Separation of Magnetic Nanoparticles.

Realizing the full potential of magnetic nanoparticles (MNPs) in nanomedicine requires the optimization of their physical and chemical properties. Elucidation of the effects of these properties on clinical diagnostic or therapeutic properties, however, requires the synthesis or purification of homogenous samples, which has proved to be difficult. While initial simulations indicated that size-selective separation could be achieved by flowing magnetic nanoparticles through a magnetic field, subsequent in vitro experiments were unable to reproduce the predicted results. Magnetic field-flow fractionation, however, was found to be an effective method for the separation of polydisperse suspensions of iron oxide nanoparticles with diameters greater than 20 nm. While similar methods have been used to separate magnetic nanoparticles before, no previous work has been done with magnetic nanoparticles between 20 and 200 nm. Both transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis were used to confirm the size of the MNPs. Further development of this work could lead to MNPs with the narrow size distributions necessary for their in vitro and in vivo optimization.

Toward Accumulation of Magnetic Nanoparticles into Tissues of Small Porosity.

Magnetic concentration of drug-laden magnetic nanoparticles has been proven to increase the delivery efficiency of treatment by 2-fold. In these techniques, particles are concentrated by the presence of a magnetic source that delivers a very high magnetic field and a strong magnetic field gradient. We have found that such magnetic conditions cause even 150 nm particles to aggregate significantly into assemblies that exceed several micrometers in length within minutes. Such assembly sizes exceed the effective intercellular pore size of tumor tissues preventing these drug-laden magnetic nanoparticles from reaching their target sites. We demonstrate that by using dynamic magnetic fields instead, we can break up these magnetic nanoparticles while simultaneously concentrating them at target sites. The dynamic fields we investigate involve precessing the field direction while maintaining a field gradient. Manipulating the field direction drives the particles into attractive and repulsive configurations that can be tuned to assemble or disassemble these particle clusters. Here, we develop a simple analytic model to describe the kinetic thresholds of disassembly and we compare both experimental and numerical results of magnetic particle suspensions subjected to dynamic fields. Finally we apply these methods to demonstrate penetration in a porous scaffold with a similar pore size to that expected of a tumor tissue.

PTD-Modified ATTEMPTS for Enhanced Toxin-based Cancer Therapy: An In Vivo Proof-of-Concept Study.

PURPOSE: To investigate the feasibility of applying PTD-modified ATTEMPTS (Antibody Targeted Triggered Electrically Modified Prodrug-Type Strategy) for enhanced toxin therapy for the treatment of cancer. METHODS: A heparin-functionalized murine anti-CEA monoclonal antibody (mAb), T84.66-heparin (T84.66-Hep), was chemically synthesized and characterized for specific binding to CEA overexpressed cells. The T84.66-Hep was then applied to the PTD-modified ATTEMPTS approach and the crucial features of the drug delivery system (DDS), 'antibody targeting' and 'heparin/protamine-based prodrug', were evaluated in vitro to examine whether it could selective delivery a PTD-modified toxin, recombinant TAT-gelonin chimera (TAT-Gel), to CEA high expression cancer cells (LS174T). Furthermore, the feasibility of the drug delivery system (DDS) was assessed in vivo by biodistribution and efficacy studies using LS174T s.c. xenograft tumor bearing mice. RESULTS: T84.66-Hep displayed specific binding, but limited internalization (35% after 48 h incubation) to CEA high expression LS174T cells over low expression HCT116 cells. When mixed together with TAT-Gel, the T84.66-Hep formed a strong yet reversible complex. This complex formation provided an effective means of active tumor targeting of TAT-Gel, by 1) directing the TAT-Gel to CEA overexpressed tumor cells and 2) preventing nonspecific cell transduction to non-targeted normal cells. The cell transduction of TAT-Gel could, however, be efficiently reversed by addition of protamine. Feasibility of in vivo tumor targeting and "protamine-induced release" of TAT-Gel from the T84.66-Hep counterpart was confirmed by biodistribution and preliminary efficacy studies. CONCLUSIONS: This study successfully demonstrated in vitro and in vivo the applicability of PTD-modified ATTEMPTS for toxin-based cancer therapy.

Scaffold materials from glycosylated and PEGylated bovine serum albumin.

Bovine serum albumin has been PEGylated and glycosylated to create mimetic materials for the extracellular matrix (ECM) with potential tissue engineering applications. Different surfaces for cell adhesion were achieved by crosslinking the initial albumin product and forming either a coating or a sponge-like three-dimensional morphology to mimic the mesh structure of natural ECM. The biocompatibility of the albumin matrix with mammalian cells was evaluated using cell culture assays with NIH 3T3 cells. The results indicated that glycoprotein composition and specific morphology of the assembly can improve the cell growth environment. These ECM mimetic structures might eventually be considered to serve as alternatives for the more expensive collagen and elastin based ECM substances currently in use in tissue engineering.

Effects of Iron-Oxide Nanoparticle Surface Chemistry on Uptake Kinetics and Cytotoxicity in CHO-K1 Cells.

Superparamagnetic iron-oxide nanoparticles (SPIONs) show great promise for multiple applications in biomedicine. While a number of studies have examined their safety profile, the toxicity of these particles on reproductive organs remains uncertain. The goal of this study was to evaluate the cytotoxicity of starch-coated, aminated, and PEGylated SPIONs on a cell line derived from Chinese Hamster ovaries (CHO-K1 cells). We evaluated the effect of particle diameter (50 and 100 nm) and polyethylene glycol (PEG) chain length (2k, 5k and 20k Da) on the cytotoxicity of SPIONs by investigating cell viability using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine B (SRB) assays. The kinetics and extent of SPION uptake by CHO-K1 cells was also studied, as well as the resulting generation of intracellular reactive oxygen species (ROS). Cell toxicity profiles of SPIONs correlated strongly with their cellular uptake kinetics, which was strongly dependent on surface properties of the particles. PEGylation caused a decrease in both uptake and cytotoxicity compared to aminated SPIONs. Interestingly, 2k Da PEG-modifed SPIONs displayed the lowest cellular uptake and cytotoxicity among all studied particles. These results emphasize the importance of surface coatings when engineering nanoparticles for biomedical applications.

Cell penetrating peptides and the mechanisms for intracellular entry.

A major thrust in the biomedical and pharmaceutical industries is to develop diagnostic and therapeutic tools that have significantly improved selectivity and specificity compared to the current state-of-the-art. This has driven much of the effort to look at molecules and materials that are significantly larger than the traditional small molecule agents. Due to size restrictions, however, many of these materials are unable to penetrate the cell membrane and gain access to the intracellular components on which they exert their action. The relatively recent discovery of cell penetrating peptides (CPP) provides a powerful tool that has enabled the intracellular delivery of these materials. While a variety of proteins, DNA, polymers and even nanoparticles have been successfully delivered into cells, there still remains some debate as to the mechanism of entry utilized by the CPPs. In this review, we provide a brief outline of the different potential mechanisms for cellular uptake of CPPs.

Cell-penetrating peptides: achievements and challenges in application for cancer treatment.

One of the major hurdles to cure cancer lies in the low potency of currently available drugs, which could eventually be solved by using more potent therapeutic macromolecules, such as proteins or genes. However, although these macromolecules possess greater potency inside the cancer cells, the barely permeable cell membrane remains a formidable barrier to exert their efficacy. A widely used strategy is to use cell penetrating peptides (CPPs) to improve their intracellular uptake. Since the discovery of the first CPP, numerous CPPs have been derived from natural or synthesized products. Both in vitro and in vivo studies have demonstrated that those CPPs are highly efficient in transducing cargoes into almost all cell types. Therefore, to date, CPPs have been widely used for intracellular delivery of various cargoes, including peptides, proteins, genes, and even nanoparticles. In addition, recently, based on the successes of CPPs in cellular studies, their applications in vivo have been actively pursued. This review will focus on the advanced applications of CPP-based in vivo delivery of therapeutics (e.g., small molecule drugs, proteins, and genes). In addition, we will highlight certain updated applications of CPPs for intracellular delivery of nanoparticulate drug carriers, as well as several "smart" strategies for tumor targeted delivery of CPP-cargoes.

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.

Magnetic tumor targeting of ß-glucosidase immobilized iron oxide nanoparticles.

Directed enzyme/prodrug therapy (DEPT) has promising application for cancer therapy. However, most current DEPT strategies face shortcomings such as the loss of enzyme activity during preparation, low delivery and transduction efficiency in vivo and difficultly of monitoring. In this study, a novel magnetic directed enzyme/prodrug therapy (MDEPT) was set up by conjugating ß-glucosidase (ß-Glu) to aminated, starch-coated, iron oxide magnetic iron oxide nanoparticles (MNPs), abbreviated as ß-Glu-MNP, using glutaraldehyde as the crosslinker. This ß-Glu-MNP was then characterized in detail by size distribution, zeta potential, FTIR spectra, TEM, SQUID and magnetophoretic mobility analysis. Compared to free enzyme, the conjugated ß-Glu on MNPs retained 85.54% ± 6.9% relative activity and showed much better temperature stability. The animal study results showed that ß-Glu-MNP displays preferable pharmacokinetics characteristics in relation to MNPs. With an adscititious magnetic field on the surface of a tumor, a significant quantity of ß-Glu-MNP was selectively delivered into a subcutaneous tumor of a glioma-bearing mouse. Remarkably, the enzyme activity of the delivered ß-Glu in tumor lesions showed as high as 20.123±5.022 mU g(-1) tissue with 2.14 of tumor/non-tumor ß-Glu activity.

Chemically and biologically synthesized CPP-modified gelonin for enhanced anti-tumor activity.

The ineffectiveness of small molecule drugs against cancer has generated significant interest in more potent macromolecular agents. Gelonin, a plant-derived toxin that inhibits protein translation, has attracted much attention in this regard. Due to its inability to internalize into cells, however, gelonin exerts only limited tumoricidal effect. To overcome this cell membrane barrier, we modified gelonin, via both chemical conjugation and genetic recombination methods, with low molecular weight protamine (LMWP), a cell-penetrating peptide (CPP) which was shown to efficiently ferry various cargoes into cells. Results confirmed that gelonin-LMWP chemical conjugate (cG-L) and recombinant gelonin-LMWP chimera (rG-L) possessed N-glycosidase activity equivalent to that of unmodified recombinant gelonin (rGel); however, unlike rGel, both gelonin-LMWPs were able to internalize into cells. Cytotoxicity studies further demonstrated that cG-L and rG-L exhibited significantly improved tumoricidal effects, with IC50 values being 120-fold lower than that of rGel. Moreover, when tested against a CT26 s.c. xenograft tumor mouse model, significant inhibition of tumor growth was observed with rG-L doses as low as 2 µg/tumor, while no detectable therapeutic effects were seen with rGel at 10-fold higher doses. Overall, this study demonstrated the potential of utilizing CPP-modified gelonin as a highly potent anticancer drug to overcome limitations of current chemotherapeutic agents.

The use of low molecular weight protamine chemical chimera to enhance monomeric insulin intestinal absorption.

Although oral delivery of insulin offers a number of unmatched advantages, it nevertheless is beset by the poor permeability of insulin molecules through the epithelial cell membranes of the intestinal mucosal layer. We previously reported the development of low molecular weight protamine (LMWP) as a non-toxic yet potent cell-penetrating peptide, of which via covalent linkage was capable of translocating protein cargos through the membranes of almost all cell types. It is therefore hypothesized that LMWP could be practically employed as a safe and effective tool to deliver insulin across the intestinal mucosal membrane, thereby augmenting its absorption through the GI tract. However, formulating 1:1 monomeric insulin/LMWP conjugate presents a tall order of challenge, as the acidic insulin and basic LMWP would automatically form tight aggregates through electrostatic interactions. In this paper, we developed an innovative conjugation strategy to solve this problem, by using succinimidyl-[(N-maleimidopropionamido)-polyethyleneglycol] ester (NHS-PEG-MAL) as an intermediate cross-linker during the coupling process. Both SDS-PAGE and MALDI-TOF mass spectroscopy confirmed the formation of a homogenous, monomeric (1:1 ratio) insulin/LMWP conjugate without encountering the conventional problem of substrate aggregation. Cell culture studies demonstrated that transport of the Insulin-PEG-LMWP conjugate across the intestinal mucosal monolayer was augmented by almost five-folds compared to native insulin. Furthermore, results from the in situ loop absorption tests in rats showed that systemic pharmacological bioavailability of insulin was significantly enhanced after its conjugation with LMWP. Overall, the presented chemical conjugation with LMWP could offer a reliable and safe means to improve the intestinal permeability of therapeutic peptides/proteins, shedding light of the possibility for their effective oral delivery.