Tuning protein half-life in mouse using sequence-defined biopolymers functionalized with lipids.

The use of biologics in the treatment of numerous diseases has increased steadily over the past decade due to their high specificities, low toxicity, and limited side effects. Despite this success, peptide- and protein-based drugs are limited by short half-lives and immunogenicity. To address these challenges, we use a genomically recoded organism to produce genetically encoded elastin-like polypeptide-protein fusions containing multiple instances of para-azidophenylalanine (pAzF). Precise lipidation of these pAzF residues generated a set of sequence-defined synthetic biopolymers with programmable binding affinity to albumin without ablating the activity of model fusion proteins, and with tunable blood serum half-lives spanning 5 to 94% of albumin's half-life in a mouse model. Our findings present a proof of concept for the use of genetically encoded bioorthogonal conjugation sites for multisite lipidation to tune protein stability in mouse serum. This work establishes a programmable approach to extend and tune the half-life of protein or peptide therapeutics and a technical foundation to produce functionalized biopolymers endowed with programmable chemical and biophysical properties with broad applications in medicine, materials science, and biotechnology.

Antibody-functionalized polymer nanoparticle leading to memory recovery in Alzheimer's disease-like transgenic mouse model.

Alzheimer's disease (AD) is a neurodegenerative disorder related, in part, to the accumulation of amyloid-ß peptide (Aß) and especially the Aß peptide 1-42 (Aß1-42). The aim of this study was to design nanocarriers able to: (i) interact with the Aß1-42 in the blood and promote its elimination through the "sink effect" and (ii) correct the memory defect observed in AD-like transgenic mice. To do so, biodegradable, PEGylated nanoparticles were surface-functionalized with an antibody directed against Aß1-42. Treatment of AD-like transgenic mice with anti-Aß1-42-functionalized nanoparticles led to: (i) complete correction of the memory defect; (ii) significant reduction of the Aß soluble peptide and its oligomer level in the brain and (iii) significant increase of the Aß levels in plasma. This study represents the first example of Aß1-42 monoclonal antibody-decorated nanoparticle-based therapy against AD leading to complete correction of the memory defect in an experimental model of AD.

Challenges and opportunities in the delivery of cancer therapeutics: update on recent progress.

Global cancer prevalence has continuously increased in the last decades despite substantial progress achieved for patient care. Cancer is no longer recognized as a singular disease but as a plurality of different ones, leading to the important choice of the drug administration route and promoting the development of novel drug-delivery systems (DDS). Due to their structural diversity, therapeutic cancer drugs present specific challenges in physicochemical properties that can adversely affect their efficacy and toxicity profile. These challenges are addressed by innovative DDS to improve bioavailability, pharmacokinetics and biodistribution profiles. Here, we define the drug delivery challenges related to oral, intravenous, subcutaneous or alternative routes of administration, and review innovative DDS, marketed or in development, that answer those challenges.

A "top-down" approach to actuate poly(amine-co-ester) terpolymers for potent and safe mRNA delivery.

Gene delivery is known to be a complicated multi-step biological process. It has been observed that subtle differences in the structure and properties of polymeric materials used for gene delivery can lead to dramatic differences in transfection efficiency. Therefore, screening of properties is pivotal to optimizing the polymer. So far, most polymeric materials are built in a "bottom-up" manner, i.e. synthesized from monomers that allow modification of polymer composition or structural factors. With this method, we previously synthesized and screened a library of biodegradable poly(amine-co-ester) (PACE) terpolymers for optimized DNA delivery. However, it can be tedious and time consuming to synthesize a polymer library for screening, particularly when small changes of a factor need to be tested, when multiple factors are involved, and when the effects of different factors are synergistic. In the present work, we evaluate the potential of PACE to deliver mRNA. After observing that mRNA transfection efficiency was highly dependent on both end group composition and molecular weight (MW) of PACE in a synergistic manner, we developed a "top-down" process we called actuation, to simultaneously vary these two factors. Some of the actuated PACE (aPACE) materials presented superior mRNA delivery properties compared to regular PACE, with up to a 106-fold-increase in mRNA transfection efficiency in vitro. Moreover, when aPACE was used to deliver mRNA coding for erythropoietin (EPO) in vivo, it produced high levels of EPO in the blood for up to 48 h without inducing systemic toxicity. This polymer constitutes a new delivery vehicle for mRNA-based treatments that provides safe yet potent protein production.

Improved threshold selection for the determination of volume of distribution of nanoparticles administered by convection-enhanced delivery.

Nanotechnology, in conjunction with convection-enhanced delivery (CED), has gained traction as a promising method to treat many debilitating neurological diseases, including gliomas. One of the key parameters to evaluate the effectiveness of delivery is the volume of distribution (Vd) of nanoparticles within the brain parenchyma. Measurements of Vd are commonly made using fluorescent reporter systems. However, reported analyses lack accurate and robust methods for determining Vd. Current methods face the problems of varying background intensities between images, high intensity aggregates that can shift intensity distributions, and faint residual backgrounds that can occur as artifacts of fluorescent imaging. These problems can cause inaccurate results to be reported when a percentage of the maximum intensity is set as the threshold value. Here we show an implementation of Otsu's method more reliably selects accurate threshold values than the fixed-threshold method. We also introduce a goodness of fit value η that quantifies the appropriateness of using Otsu's method to calculate Vd. Adoption of Otsu's method and reporting of η may help standardize fluorescent image analysis of nanoparticles administered by convection-enhanced delivery.

Surface chemistry governs cellular tropism of nanoparticles in the brain.

Nanoparticles are of long-standing interest for the treatment of neurological diseases such as glioblastoma. Most past work focused on methods to introduce nanoparticles into the brain, suggesting that reaching the brain interstitium will be sufficient to ensure therapeutic efficacy. However, optimized nanoparticle design for drug delivery to the central nervous system is limited by our understanding of their cellular deposition in the brain. Here, we investigated the cellular fate of poly(lactic acid) nanoparticles presenting different surface chemistries, after administration by convection-enhanced delivery. We demonstrate that nanoparticles with 'stealth' properties mostly avoid internalization by all cell types, but internalization can be enhanced by functionalization with bio-adhesive end-groups. We also show that association rates measured in cultured cells predict the extent of internalization of nanoparticles in cell populations. Finally, evaluating therapeutic efficacy in an orthotopic model of glioblastoma highlights the need to balance significant uptake without inducing adverse toxicity.

Distribution of polymer nanoparticles by convection-enhanced delivery to brain tumors.

Glioblastoma multiforme (GBM) is a fatal brain tumor characterized by infiltration beyond the margins of the main tumor mass and local recurrence after surgery. The blood-brain barrier (BBB) poses the most significant hurdle to brain tumor treatment. Convection-enhanced delivery (CED) allows for local administration of agents, overcoming the restrictions of the BBB. Recently, polymer nanoparticles have been demonstrated to penetrate readily through the healthy brain when delivered by CED, and size has been shown to be a critical factor for nanoparticle penetration. Because these brain-penetrating nanoparticles (BPNPs) have high potential for treatment of intracranial tumors since they offer the potential for cell targeting and controlled drug release after administration, here we investigated the intratumoral CED infusions of PLGA BPNPs in animals bearing either U87 or RG2 intracranial tumors. We demonstrate that the overall volume of distribution of these BPNPs was similar to that observed in healthy brains; however, the presence of tumors resulted in asymmetric and heterogeneous distribution patterns, with substantial leakage into the peritumoral tissue. Together, our results suggest that CED of BPNPs should be optimized by accounting for tumor geometry, in terms of location, size and presence of necrotic regions, to determine the ideal infusion site and parameters for individual tumors.

PEGylated squalenoyl-gemcitabine nanoparticles for the treatment of glioblastoma.

New treatments for glioblastoma multiforme (GBM) are desperately needed, as GBM prognosis remains poor, mainly due to treatment resistance, poor distribution of therapeutics in the tumor tissue, and fast metabolism of chemotherapeutic drugs in the brain extracellular space. Convection-enhanced delivery (CED) of nanoparticles (NPs) has been shown to improve the delivery of chemotherapeutic drugs to the tumor bed, providing sustained release, and enhancing survival of animals with intracranial tumors. Here we administered gemcitabine, a nucleoside analog used as a first line treatment for a wide variety of extracranial solid tumors, within squalene-based NPs using CED, to overcome the above-mentioned challenges of GBM treatment. Small percentages of poly(ethylene) glycol (PEG) dramatically enhanced the distribution of squalene-gemcitabine nanoparticles (SQ-Gem NPs) in healthy animals and tumor-bearing animals after administration by CED. When tested in an orthotopic model of GBM, SQ-Gem-PEG NPs demonstrated significantly improved therapeutic efficacy compared to free gemcitabine, both as a chemotherapeutic drug and as a radiosensitizer. Furthermore, MR contrast agents were incorporated into the SQ-Gem-PEG NP formulation, providing a way to non-invasively track the NPs during infusion.

Pharmacokinetics, biodistribution and metabolism of squalenoyl adenosine nanoparticles in mice using dual radio-labeling and radio-HPLC analysis.

Adenosine is a pleiotropic endogenous nucleoside with potential neuroprotective pharmacological activity. However, clinical use of adenosine is hampered by its extremely fast metabolization. To overcome this limitation, we recently developed a new squalenoyl nanomedicine of adenosine [Squalenoyl-Adenosine (SQAd)] by covalent linkage of this nucleoside to the squalene, a natural lipid. The resulting nanoassemblies (NAs) displayed a dramatic pharmacological activity both in cerebral ischemia and spinal cord injury pre-clinical models. The aim of the present study was to investigate the plasma profile and tissue distribution of SQAd NAs using both Squalenoyl-[(3)H]-Adenosine NAs and [(14)C]-Squalenoyl-Adenosine NAs as respective tracers of adenosine and squalene moieties of the SQAd bioconjugate. This study was completed by radio-HPLC analysis allowing to determine the metabolization profile of SQAd. We report here that SQAd NAs allowed a sustained circulation of adenosine under its prodrug form (SQAd) for at least 1h after intravenous administration, when free adenosine was metabolized within seconds after injection. Moreover, the squalenoylation of adenosine and its formulation as NAs also significantly modified biodistribution, as SQAd NAs were mainly captured by the liver and spleen, allowing a significant release of adenosine in the liver parenchyma. Altogether, these results suggest that SQAd NAs provided a reservoir of adenosine into the bloodstream which may explain the previously observed neuroprotective efficacy of SQAd NAs against cerebral ischemia and spinal cord injury.

Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injury.

There is an urgent need to develop new therapeutic approaches for the treatment of severe neurological trauma, such as stroke and spinal cord injuries. However, many drugs with potential neuropharmacological activity, such as adenosine, are inefficient upon systemic administration because of their fast metabolization and rapid clearance from the bloodstream. Here, we show that conjugation of adenosine to the lipid squalene and the subsequent formation of nanoassemblies allows prolonged circulation of this nucleoside, providing neuroprotection in mouse stroke and rat spinal cord injury models. The animals receiving systemic administration of squalenoyl adenosine nanoassemblies showed a significant improvement of their neurologic deficit score in the case of cerebral ischaemia, and an early motor recovery of the hindlimbs in the case of spinal cord injury. Moreover, in vitro and in vivo studies demonstrated that the nanoassemblies were able to extend adenosine circulation and its interaction with the neurovascular unit. This Article shows, for the first time, that a hydrophilic and rapidly metabolized molecule such as adenosine may become pharmacologically efficient owing to a single conjugation with the lipid squalene.