Stefano Menegatti

Platelet-like Nanoparticles: Mimicking Shape, Flexibility, and Surface Biology of Platelets To Target Vascular Injuries

Targeted delivery of therapeutic and imaging agents in the vascular compartment represents a significant hurdle in using nanomedicine for treating hemorrhage, thrombosis, and atherosclerosis. While several types of nanoparticles have been developed to meet this goal, their utility is limited by poor circulation, limited margination, and minimal targeting. Platelets have an innate ability to marginate to the vascular wall and specifically interact with vascular injury sites. These platelet functions are mediated by their shape, flexibility, and complex surface interactions. Inspired by this, we report the design and evaluation of nanoparticles that exhibit platelet-like functions including vascular injury site-directed margination, site-specific adhesion, and amplification of injury site-specific aggregation. Our nanoparticles mimic four key attributes of platelets, (i) discoidal morphology, (ii) mechanical flexibility, (iii) biophysically and biochemically mediated aggregation, and (iv) heteromultivalent presentation of ligands that mediate adhesion to both von Willebrand Factor and collagen, as well as specific clustering to activated platelets. Platelet-like nanoparticles (PLNs) exhibit enhanced surface-binding compared to spherical and rigid discoidal counterparts and site-selective adhesive and platelet-aggregatory properties under physiological flow conditions in vitro. In vivo studies in a mouse model demonstrated that PLNs accumulate at the wound site and induce ∼65% reduction in bleeding time, effectively mimicking and improving the hemostatic functions of natural platelets. We show that both the biochemical and biophysical design parameters of PLNs are essential in mimicking platelets and their hemostatic functions. PLNs offer a nanoscale technology that integrates platelet-mimetic biophysical and biochemical properties for potential applications in injectable synthetic hemostats and vascularly targeted payload delivery.

Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media.

Previous work has reported on the identification and characterization of the hexapeptide ligands HWRGWV, HYFKFD, and HFRRHL for the affinity capture of IgG through specific binding to its Fc fragment. This paper addresses issues related to the successful application of these ligands, on a commercial methacrylate chromatographic resin, for the purification of IgG from mammalian cell culture fluids. The concentrations of sodium chloride and sodium caprylate in the binding buffer were optimized to maximize the purity and yield of IgG upon elution. Screening of several regeneration conditions found that either 2M guanidine-HCl or a combination of 0.85% phosphoric acid followed by 2M urea resulted in complete recovery of the IgG adsorption capacity and that the column could be reused over many cycles. The hexapeptide ligands were used for the purification of humanized and chimeric monoclonal antibodies from two commercial CHO cell culture fluids. The chimeric MAb of IgG1 subclass was purified using the HWRGWV resin whereas the humanized MAb of IgG4 subclass was purified using the HWRGWV, HYFKFD and HFRRHL resins. The purities and yields obtained for both the MAbs were found to be higher than 94% and 85% respectively. These results compare well with the yields and purities obtained using Protein G columns. The residual DNA and host cell protein reduction obtained by the HWRGWV resin was in the range of 4 log reduction value (LRV) and 2 LRV respectively, comparable to those reported for Protein A resins. The dynamic binding capacity of all three peptide resins for the humanized monoclonal antibody was in the range of 20mg/mL.

Peptides as skin penetration enhancers: mechanisms of action.

Skin penetrating peptides (SPPs) have garnered wide attention in recent years and emerged as a simple and effective noninvasive strategy for macromolecule delivery into the skin. Although SPPs have demonstrated their potential in enhancing skin delivery, they are still evolving as a new class of skin penetration enhancers. Detailed studies elucidating their mechanisms of action are still lacking. Using five SPPs (SPACE peptide, TD-1, polyarginine, a dermis-localizing peptide and a skin penetrating linear peptide) and a model hydrophobic macromolecule (Cyclosporine A, CsA), herein we provide a mechanistic understanding of SPPs. To evaluate the mechanism and safety of SPPs, their effects on skin lipids, proteins and keratinocyte cells were evaluated. Three SPPs (SPACE, Polyarginine and TD-1) significantly enhanced CsA penetration into the skin. SPPs did not alter the skin lipid barrier as measured by skin resistance, transepidermal water loss (TEWL) and Fourier transform infrared (FTIR) spectroscopic analysis. In contrast, SPPs interacted with skin proteins and induced changes in skin protein secondary structures (α-helices, β-sheet, random coils and turns), as evaluated by FTIR analysis and confirmed by in-silico docking. SPPs enhanced CsA skin penetration, via a transcellular pathway, enhancing its partitioning into keratin-rich corneocytes through concurrent binding of SPP with keratin and CsA. Interaction between SPP and keratin best correlated with measured CsA skin transport. Many SPPs appeared to be safe as shown by negligible effect on skin integrity, nominal skin irritation potential and cytotoxicity. Among the peptides tested, SPACE peptide was found to be least toxic to keratinocytes, and among the most effective at delivering CsA into the skin.

mRNA display selection and solid-phase synthesis of Fc-binding cyclic peptide affinity ligands

Cyclic peptides are attractive candidates for synthetic affinity ligands due to their favorable properties, such as resistance to proteolysis, and higher affinity and specificity relative to linear peptides. Here we describe the discovery, synthesis and characterization of novel cyclic peptide affinity ligands that bind the Fc portion of human Immunoglobulin G (IgG; hFc). We generated an mRNA display library of cyclic pentapeptides wherein peptide cyclization was achieved with high yield and selectivity, using a solid‐phase crosslinking reaction between two primary amine groups, mediated by a homobifunctional linker. Subsequently, a pool of cyclic peptide binders to hFc was isolated from this library and chromatographic resins incorporating the selected cyclic peptides were prepared by on‐resin solid‐phase peptide synthesis and cyclization. Significantly, this approach results in resins that are resistant to harsh basic conditions of column cleaning and regeneration. Further studies identified a specific cyclic peptide—cyclo[Link‐M‐WFRHY‐K]—as a robust affinity ligand for purification of IgG from complex mixtures. The cyclo[Link‐M‐WFRHY‐K] resin bound selectively to the Fc fragment of IgG, with no binding to the Fab fragment, and also bound immunoglobulins from a variety of mammalian species. Notably, while the recovery of IgG using the cyclo[Link‐M‐WFRHY‐K] resin was comparable to a Protein A resin, elution of IgG could be achieved under milder conditions (pH 4 vs. pH 2.5). Thus, cyclo[Link‐M‐WFRHY‐K] is an attractive candidate for developing a cost‐effective and robust chromatographic resin to purify monoclonal antibodies (mAbs). Finally, our approach can be extended to efficiently generate and evaluate cyclic peptide affinity ligands for other targets of interest. Biotechnol. Bioeng. 2013; 110: 857–870.

Synergistic antitumor activity of camptothecin–doxorubicin combinations and their conjugates with hyaluronic acid

Combinations of topoisomerase inhibitors I and II have been found to synergistically inhibit cancer cell growth in vitro, yet clinical studies of these types of combinations have not progressed beyond phase II trials. The results of clinical combinations of topoisomerase (top) I and II inhibitors typically fall within one of two categories: little to no improvement in therapeutic efficacy, or augmented toxicity compared to the single drug counterparts. Hence, despite the promising activity of top I and II inhibitor combinations in vitro, their clinical applicability has not been realized. Here, we report the use of polymer-drug conjugates as a means to co-deliver synergistic doses of top I and II inhibitors camptothecin (CPT) and doxorubicin (DOX) to tumors in vivo in a 4T1 breast cancer model. At specific molar ratios, DOX and CPT were found to be among the most synergistic combinations reported to date, with combination indices between 0.01 and 0.1. The identified optimal ratios were controllably conjugated to hyaluronic acid, and elicited significant tumor reduction of murine 4T1 breast cancer model when administered intravenously. This study elucidates a method to identify synergistic drug combinations and translate them to in vivo by preserving the synergistic ratio via conjugation to a carrier polymer, thus opening a promising approach to translate drug combinations to clinically viable treatment regimens.

Process for purification of monoclonal antibody expressed in transgenic Lemna plant extract using dextran-coated charcoal and hexamer peptide affinity resin.

The production of therapeutic proteins using transgenic plants offers several advantages, including low production cost, absence of human pathogens, presence of glycosylation mechanisms, and the ability to fold complex therapeutic proteins into their proper conformation. However, impurities such as phenolic compounds and pigments encountered during purification are quite different from those faced during purification from mammalian cell culture supernatants. This paper deals with the development of a pretreatment and affinity separation process for the purification of a monoclonal antibody from transgenic Lemna plant extract. A pretreatment step is described using dextran-coated charcoal for the removal of pigments and phenolic compounds without reducing the antibody concentration. Then, the peptide affinity ligand HWRGWV coupled to a commercial polymethacrylate resin is used for the capture and purification of MAb from the pretreated plant extract. The final yield and purity of the MAb obtained were 90% and 96% respectively. The performance of the hexamer peptide resin after the pretreatment step was found to be similar to that obtained with a commercial Protein A resin.

Purification of polyclonal antibodies from Cohn fraction II + III, skim milk, and whey by affinity chromatography using a hexamer peptide ligand.

HWRGWV, a peptide that binds specifically to the Fc fragment of human immunoglobulin G (IgG), was used for the purification of IgG from Cohn fraction II + III of human plasma and from bovine skim milk and whey. The concentration of sodium chloride and sodium caprylate in the binding buffer as well as the pH of the elution buffer were optimized to achieve high IgG yield and purity. Under optimized conditions, IgG was recovered from plasma fractions with yield and purity up to 84% and 95%, respectively. IgG was also purified from skim milk with 74% yield and 92% purity and from whey with 85% yield and 93% purity. Purification experiments were also performed with Protein A resin and the results were found to be similar to those obtained with the peptide adsorbent.

Alkaline-stable peptide ligand affinity adsorbents for the purification of biomolecules.

A strategy of modification of resin surface chemistry is presented to produce hydrophilic peptide-based alkaline-stable affinity adsorbents for the purification of biopharmaceuticals from complex media. In this work, the peptide-based affinity adsorbent HWRGWV-Toyopearl resin for the purification of IgG is presented as an example. When prepared by direct peptide synthesis on the chromatographic matrix, the peptide-based resin showed lability under alkaline conditions. In fact, the regeneration with aqueous 0.1 M NaOH caused the leaching of 40% of the peptide ligand, resulting in a decrease of IgG yield from 85% to 23%. It was found that the ligand leaching was caused by the coupling of a significant amount of peptide by alkaline-labile ester bonds. A method was designed to prevent the formation of ester bonds and allow the synthesis of the ligand exclusively on alkaline-stable bonds. The method consists in activating the hydrophilic base resin, blocking the hydroxyl groups responsible for alkaline lability and performing the peptide synthesis exclusively via alkaline-stable amide bonds. Repeated cycles of IgG purification from a cell culture medium were performed, each followed by cleaning with aqueous NaOH (0.1 M, 0.5 M and 1 M). The IgG yield decreased from 91% to 85% after 200 purification cycles with 0.1 M NaOH. However, the IgG purity remained almost constant at around 95% based on SDS-PAGE analysis. The procedure presented is rapid, efficient and inexpensive and does not require any equipment other than the conventional instrumentation for peptide synthesis. The method also has a broad application since it is valid for any peptide ligand identified for the purification of a biopharmaceutical target.

DAFODIL: A novel liposome-encapsulated synergistic combination of doxorubicin and 5FU for low dose chemotherapy

PEGylated liposomes have transformed chemotherapeutic use of doxorubicin by reducing its cardiotoxicity; however, it remains unclear whether liposomal doxorubicin is therapeutically superior to free doxorubicin. Here, we demonstrate a novel PEGylated liposome system, named DAFODIL (Doxorubicin And 5-Flurouracil Optimally Delivered In a Liposome) that inarguably offers superior therapeutic efficacies compared to free drug administrations. Delivery of synergistic ratios of this drug pair led to greater than 90% reduction in tumor growth of murine 4T1 mammary carcinoma in vivo. By exploiting synergistic ratios, the effect was achieved at remarkably low doses, far below the maximum tolerable drug doses. Our approach re-invents the use of liposomes for multi-drug delivery by providing a chemotherapy vehicle which can both reduce toxicity and improve therapeutic efficacy. This methodology is made feasible by the extension of the ammonium-sulfate gradient encapsulation method to nucleobase analogues, a liposomal entrapment method once conceived useful only for anthracyclines. Therefore, our strategy can be utilized to efficiently evaluate various chemotherapy combinations in an effort to translate more effective combinations into the clinic.

Reversible cyclic peptide libraries for the discovery of affinity ligands.

A novel strategy is presented for the identification of cyclic peptide ligands from combinatorial libraries of reversible cyclic depsipeptides. A method for the solid-phase synthesis of individual cyclic depsipeptides and combinatorial libraries of these compounds is proposed, which employs lactic acid (Lact) and the dipeptide ester (Nα-Ac)-Ser(Ala)- as linkers for dilactonization. Upon alkaline treatment of the beads selected by screening a model library, the cyclic depsipeptides are linearized and released from the solid support to the liquid phase, to be sequenced via single-step tandem mass spectrometry (MS/MS). The protocol presented for library synthesis provides for wide structural diversity. Two model sequences, VVWVVK and AAWAAR, were chosen to present different structural examples for depsipeptide libraries and demonstrate the process of sequence determination by mass spectrometry. Further, a case study using the IgG binding cyclic depsipeptide cyclo[(Nα-Ac)-S(A)-RWHYFK-Lact-E] is presented to demonstrate the process of library screening and sequence determination on the selected beads. Finally, a method is shown for synthesis of the irreversible cyclic peptide corresponding to the proposed depsipeptide structure, to make the ligand stable to the aqueous acid and alkaline conditions encountered in affinity chromatographic applications. The cyclic peptide ligand was synthesized on a poly(methacrylate) resin and used for chromatographic binding of the target IgG.