Monodisperse and Polydisperse PEGylation of Peptides and Proteins: A Comparative Study.

Although PEGylation is widely used in biomedicine with great success, it suffers from many drawbacks, such as polydispersity, nonbiodegradability, and loss of precursor potency. Recently, the search for polyethylene glycol (PEG) substitutes has attracted considerable attention. Some of the substitutes partially address the drawbacks of PEGs, but sacrifice the "stealth" effect of PEGs and bring in new issues. Herein, we developed monodisperse oligoethylene glycol (M-OEG) polyamides over 5000 Da as biodegradable and monodisperse PEGylation (M-PEGylation) agents, which provided M-PEGylated peptides and proteins with high monodispersity and a biodegradable PEG moiety. Compared to regular PEGylated proteins with a complex "stealth" cloud of PEG, the hydrogen bond interactions between the M-OEG polyamides and proteins provided the M-PEGylated protein with a biodegradable "stealth" cloak. The monodisperse and biodegradable M-PEGylation strategy as well as the peculiar protein-M-OEG polyamide interactions may shed light on many long-lasting issues during the development of PEGylated biologic drugs, such as monodispersity, biodegradability, and tunable conformation.

Quantitatively Fine-Tuning the Physicochemical and Biological Properties of Peptidic Polymers through Monodisperse PEGylation.

In biomedicine, PEGylation is one of the most successful strategies to modify the physicochemical and biological properties of peptides, proteins, and other biomacromolecules. Because of the polydisperse nature of regular PEGs and limited PEGylation strategies, it is challenging to quantitatively fine-tune and accurately predict the properties of biomacromolecules after PEGylation. However, such fine-tuning and prediction may be crucial for their biomedical applications. Herein, some monodisperse PEGylation strategies, including backbone PEGylation, side-chain PEGylation, and highly branched PEGylation, have been developed. In a comparative fashion, the impact of PEGylation strategies and monodisperse PEG sizes on the physicochemical and biological properties, including lipophilicity, thermosensitivity, biocompatibility, plasma stability, and drug delivery capability, of peptidic polymers has been quantitatively studied. It was found that the physicochemical and biological properties of PEGylated peptidic polymers can be quantitatively fine-tuned and accurately predicted through these monodisperse PEGylation strategies. After the comparative study, a side-chain monodisperse PEGylated peptidic polymer was chosen as fluorine-19 magnetic resonance and fluorescence dual-imaging traceable drug delivery vehicle. Our study may not only promote the transformation of PEGylation from an empirical technology to a quantitative science but also shed light on the rational design of PEGylated biomaterials and pharmaceutics.

Synthesis of Branched Monodisperse Oligoethylene Glycols and 19F MRI-Traceable Biomaterials through Reductive Dimerization of Azides.

Multifunctionalized and branched M-OEGs represent valuable PEGylation agents, linkers, and scaffolds in biomedicine. However, the tedious synthesis limited their availability and application. We herein present an azide reductive dimerization method for the convenient synthesis of aza-M-OEGs and derivatives, which provides easy access to a variety of multifunctionalized and branched M-OEGs in one step. With this method, hexa-arm M-OEGs with 54 symmetrical fluorines were synthesized in two steps as a water-soluble, self-assemble, 19F MRI sensitive, and biocompatible dendritic biomaterial.

Peptidic Monodisperse PEG "combs" with Fine-Tunable LCST and Multiple Imaging Modalities.

Thermosensitive and imaging-traceable materials with fine-tunable lower critical solution temperature (LCST) around body temperature are highly valuable in biomedicine. However, such materials are rare because it is challenging to fine-tune the LCST and incorporate suitable imaging modalities. Herein, peptidic monodisperse polyethylene glycol (M-PEG) "combs" with fine-tunable LCST, "hot spot" fluorine-19 magnetic resonance imaging (19F MRI), thermoresponsive fluorescent imaging, and drug loading ability were developed through accurately programming their structures during solid phase peptide synthesis (SPPS). The easy availability, structural accuracy, biocompatibility, and versatility provide the M-PEG "combs" with promising prospects as thermoresponsive and imaging-traceable biomaterials for controlled drug delivery.

Monodisperse oligoethylene glycols modified Propofol prodrugs.

The low water solubility of Propofol resulted in complicated formulation and adverse effects during its clinical application. To improve its water solubility and maintain its anesthetic effects, Propofol prodrugs with monodisperse oligoethylene glycols as solubility enhancer were designed and synthesized. Monodisperse oligoethylene glycols enable the concise manipulation of water solubility, biocompatibility and anesthetic effects. Through the physicochemical and biological assay, a few water soluble prodrugs of Propofol were identified as promising anesthetic to overcome the drawbacks associated with Propofol.

Application of Monodisperse PEGs in Pharmaceutics: Monodisperse Polidocanols.

Polydisperse PEGs are ubiquitously used in pharmaceutical industry and biomedical research. However, the monodispersity in PEGs may play a role in the development of safe and effective PEGylated small molecular drugs. Here, to avoid the polydispersity in polidocanol, the active ingredient in a clinically used drug, a macrocyclic sulfate-based strategy for the efficient and scalable synthesis of monodisperse polidocanols, their sulfates, and their methylated derivatives, was developed. TLC and HPLC analysis indicated a complex mixture in regular polidocanol and high purities in monodisperse polidocanols and their derivatives. Assay on HUVEC, L929, and HePG2 cells showed that monodisperse polidocanols have much higher cytotoxicity and safety than that of regular polidocanol. It was found that the monodispersity of PEGs in polidocanols is crucial for achieving the optimal therapeutic results. Therefore, based on this case study, it would be beneficial to optimize PEGylated small molecular drugs with monodisperse PEGs in pharmaceutical research and development.

Amide bond-containing monodisperse polyethylene glycols beyond 10 000 Da.

Although monodisperse polyethylene glycols (M-PEGs) above 4000 Da are especially valuable in biomedical applications, their synthesis remains a long-standing challenge. To this end, a peptide-based strategy for such M-PEGs was developed. With macrocyclic sulfates as the key intermediates, a panel of oligoethylene glycol (OEG) containing ω-amino acids were prepared with high efficiency. Through solid phase peptide synthesis (SPPS), these amino acids were conveniently assembled into a series of amide bond-containing M-PEGs with high flexibility in molecular weight and amide density selection. With this strategy, an M-PEG of 10 262 Da was prepared on a gram scale and its biocompatibility was assessed in a mice model.

Highly efficient synthesis of monodisperse poly(ethylene glycols) and derivatives through macrocyclization of oligo(ethylene glycols).

A macrocyclic sulfate (MCS)-based approach to monodisperse poly(ethylene glycols) (M-PEGs) and their monofunctionalized derivatives has been developed. Macrocyclization of oligo(ethylene glycols) (OEGs) provides MCS (up to a 62-membered macrocycle) as versatile precursors for a range of monofunctionalized M-PEGs. Through iterative nucleophilic ring-opening reactions of MCS without performing group protection and activation, a series of M-PEGs, including the unprecedented 64-mer (2850 Da), can be readily prepared. Synthetic simplicity coupled with versatility of this new strategy may pave the way for broader applications of M-PEGs.

Fluorinated porphyrin-based theranostics for dual imaging and chemo-photodynamic therapy.

Convenient strategies to transform regular liposomes or nano-micelles into multifunctional theranostics would be highly valuable in cancer therapy. Herein, we developed an amphiphilic fluorinated porphyrin dendrimer as a multifunctional "add-on" module which would self-assemble onto liposomal drug delivery systems and conveniently transform the liposomes into novel theranostics. Through cancer cells and murine xenograft tumor model assays, the theranostics showed valuable fluorescence/19F magnetic resonance dual modal imaging and highly efficient chemo-photodynamic therapy. The modular strategy facilitates the convenient and standardized preparation of multifunctional theranostics.

Peptidic Monodisperse PEG "Comb" as Multifunctional "Add-On" Module for Imaging-Traceable and Thermo-Responsive Theranostics.

Monodisperse polyethylene glycols-modified (M-PEGylated) biomaterials exhibit high structural accuracy, biocompatibility, and fine-tunable physicochemical properties. To develop "smart" drug delivery systems in a controllable and convenient manner, a peptidic M-PEG "comb" with fluorinated L-lysine side chains and a fluorescent N-terminal is conveniently prepared as a 19 F magnetic resonance imaging (19 F MRI) and fluorescence dual-imaging traceable and thermo-responsive "add-on" module for liposomal theranostics in cancer therapy. The peptidic M-PEG "comb" has high biocompatibility, thermo-responsivity with a sharp lower critical solution temperature, an aggregation-induced emission fluorescence, and high 19 F MRI sensitivity. As a highly branched amphiphile, it self-assembles and firmly anchors on the doxorubicin-loaded liposomal nanoparticles, which M-PEGylates the liposomes and facilitates the thermo-responsive drug release and drug tracking with dual-imaging technologies. In a rodent xenograft model of human liver cancer HepG2 cells, the M-PEGylated liposomes exhibit long in vivo half time, low toxicity, high tumor accumulation, "hot spot" 19 F MRI, and therapeutic efficacy. With accurately programmable chemical structure, fine-tunable physicochemical and biological properties to meet the demands of diagnosis, drug delivery, and therapy, the M-PEG "comb" is promising as a versatile "add-on" module for rapid and convenient formulation of various "smart" theranostics.

Monodisperse polyethylene glycol "brushes" with enhanced lipophilicity, and thermo and plasma stability.

A convenient strategy was developed for highly branched and multifunctionalized peptidic monodisperse polyethylene glycol "brushes", which exhibit remarkable physicochemical and biological properties and potential as versatile biomaterials.

A Versatile Theranostic Nanoemulsion for Architecture-Dependent Multimodal Imaging and Dually Augmented Photodynamic Therapy.

To design a clinically translatable nanomedicine for photodynamic theranostics, the ingredients should be carefully considered. A high content of nanocarriers may cause extra toxicity in metabolism, and multiple theranostic agents would complicate the preparation process. These issues would be of less concern if the nanocarrier itself has most of the theranostic functions. In this work, a poly(ethylene glycol)-boron dipyrromethene amphiphile (PEG-F54 -BODIPY) with 54 fluorine-19 (19 F) is synthesized and employed to emulsify perfluorohexane (PFH) into a theranostic nanoemulsion (PFH@PEG-F54 -BODIPY). The as-prepared PFH@PEG-F54 -BODIPY can perform architecture-dependent fluorescence/photoacoustic/19 F magnetic resonance multimodal imaging, providing more information about the in vivo structure evolution of nanomedicine. Importantly, this nanoemulsion significantly enhances the therapeutic effect of BODIPY through both the high oxygen dissolving capability and less self-quenching of BODIPY molecules. More interestingly, PFH@PEG-F54 -BODIPY shows high level of tumor accumulation and long tumor retention time, allowing a repeated light irradiation after a single-dose intravenous injection. The "all-in-one" photodynamic theranostic nanoemulsion has simple composition, remarkable theranostic efficacy, and novel treatment pattern, and thus presents an intriguing avenue to developing clinically translatable theranostic agents.

In vivo drug tracking with 19F MRI at therapeutic dose.

Tracking drugs with 19F MRI would be beneficial for developing theranostics and optimizing drug therapy. To this end, a fluorinated dendritic amphiphile with high 19F MRI sensitivity and biocompatibility has been developed for 19F MRI tracking of doxorubicin (DOX)-loaded liposomes in mice, which may provide an effective platform to in vivo trace various drugs with 19F MRI.