Frontiers in nonviral delivery of small molecule and genetic drugs, driven by polymer chemistry and machine learning for materials informatics.

Materials informatics (MI) has immense potential to accelerate the pace of innovation and new product development in biotechnology. Close collaborations between skilled physical and life scientists with data scientists are being established in pursuit of leveraging MI tools in automation and artificial intelligence (AI) to predict material properties in vitro and in vivo. However, the scarcity of large, standardized, and labeled materials data for connecting structure-function relationships represents one of the largest hurdles to overcome. In this Highlight, focus is brought to emerging developments in polymer-based therapeutic delivery platforms, where teams generate large experimental datasets around specific therapeutics and successfully establish a design-to-deployment cycle of specialized nanocarriers. Three select collaborations demonstrate how custom-built polymers protect and deliver small molecules, nucleic acids, and proteins, representing ideal use-cases for machine learning to understand how molecular-level interactions impact drug stabilization and release. We conclude with our perspectives on how MI innovations in automation efficiencies and digitalization of data-coupled with fundamental insight and creativity from the polymer science community-can accelerate translation of more gene therapies into lifesaving medicines.

Targeted Polymersome Delivery of a Stapled Peptide for Drugging the Tumor Protein p53:BCL-2-Family Axis in Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) remains a formidable diagnosis in need of new treatment paradigms. In this work, we elucidated an opportunity for therapeutic synergy in DLBCL by reactivating tumor protein p53 with a stapled peptide, ATSP-7041, thereby priming cells for apoptosis and enhancing their sensitivity to BCL-2 family modulation with a BH3-mimetic, ABT-263 (navitoclax). While this combination was highly effective at activating apoptosis in DLBCL in vitro, it was highly toxic in vivo, resulting in a prohibitively narrow therapeutic window. We, therefore, developed a targeted nanomedicine delivery platform to maintain the therapeutic potency of this combination while minimizing its toxicity via packaging and targeted delivery of a stapled peptide. We developed a CD19-targeted polymersome using block copolymers of poly(ethylene glycol) disulfide linked to poly(propylene sulfide) (PEG-SS-PPS) for ATSP-7041 delivery into DLBCL cells. Intracellular delivery was optimized in vitro and validated in vivo by using an aggressive human DLBCL xenograft model. Targeted delivery of ATSP-7041 unlocked the ability to systemically cotreat with ABT-263, resulting in delayed tumor growth, prolonged survival, and no overt toxicity. This work demonstrates a proof-of-concept for antigen-specific targeting of polymersome nanomedicines, targeted delivery of a stapled peptide in vivo, and synergistic dual intrinsic apoptotic therapy against DLBCL via direct p53 reactivation and BCL-2 family modulation.

Effect of Charged Block Length Mismatch on Double Diblock Polyelectrolyte Complex Micelle Cores.

Polyelectrolyte complex micelles are hydrophilic nanoparticles that self-assemble in aqueous environments due to associative microphase separation between oppositely charged blocky polyelectrolytes. In this work, we employ a suite of physical characterization tools to examine the effect of charged block length mismatch on the equilibrium structure of double diblock polyelectrolyte complex micelles (D-PCMs) by mixing a diverse library of peptide and synthetic charged-neutral block polyelectrolytes with a wide range of charged block lengths (25-200 units) and chemistries. Early work on D-PCMs suggested that this class of micelles can only be formed from blocky polyelectrolytes with identical charged block lengths, a phenomenon referred to as chain length recognition. Here, we use salt annealing to create PCMs at equilibrium, which shows that chain length recognition, a longstanding hurdle to repeatable self-assembly from mismatched polyelectrolytes, can be overcome. Interestingly, D-PCM structure-property relationships display a range of values that vary systematically with the charged block lengths and chemical identity of constituent polyelectrolyte pairings and cannot be described by generalizable scaling laws. We discuss the interdependent growth behavior of the radius, ionic pair aggregation number, and density in the micelle core for three chemically distinct diblock pairings and suggest a potential physical mechanism that leads to this unique behavior. By comparing the results of these D-PCMs to the scaling laws recently developed for single diblock polyelectrolyte complex micelles (S-PCMs: diblock + homopolymer), we observe that D-PCM design schemes reduce the size and aggregation number and restrict their growth to a function of charged block length relative to S-PCMs. Understanding these favorable attributes enables more predictive use of a wider array of charged molecular building blocks to anticipate and control macroscopic properties of micelles spanning countless storage and delivery applications.

Translocation Behaviors of Synthetic Polyelectrolytes through Alpha-Hemolysin (α-HL) and Mycobacterium smegmatis Porin A (MspA) Nanopores.

DNAs have been used as probes for nanopore sensing of noncharged biomacromolecules due to its negative phosphate backbone. Inspired by this, we explored the potential of diblock synthetic polyelectrolytes as more flexible and inexpensive nanopore sensing probes by investigating translocation behaviors of PEO-b-PSS and PEO-b-PVBTMA through commonly used alpha-hemolysin (α-HL) and Mycobacterium smegmatis porin A (MspA) nanopores. Translocation recordings in different configurations of pore orientation and testing voltage indicated efficient PEO-b-PSS translocations through α-HL and PEO-b-PVBTMA translocations through MspA. This work provides insight into synthetic polyelectrolyte-based probes to expand probe selection and flexibility for nanopore sensing.

Advances in the Structural Design of Polyelectrolyte Complex Micelles.

Polyelectrolyte complex micelles (PCMs) are a unique class of self-assembled nanoparticles that form with a core of associated polycations and polyanions, microphase-separated from neutral, hydrophilic coronas in aqueous solution. The hydrated nature and structural and chemical versatility make PCMs an attractive system for delivery and for fundamental polymer physics research. By leveraging block copolymer design with controlled self-assembly, fundamental structure-property relationships can be established to tune the size, morphology, and stability of PCMs precisely in pursuit of tailored nanocarriers, ultimately offering storage, protection, transport, and delivery of active ingredients. This perspective highlights recent advances in predictive PCM design, focusing on (i) structure-property relationships to target specific nanoscale dimensions and shapes and (ii) characterization of PCM dynamics primarily using time-resolved scattering techniques. We present several vignettes from these two emerging areas of PCM research and discuss key opportunities for PCM design to advance precision medicine.

SAXS methods for investigating macromolecular and self-assembled polyelectrolyte complexes.

Polyelectrolyte complexation is driven by associative interactions between oppositely charged polyelectrolytes, resulting in formation of a macroscopic polymer dense phase and a polymer dilute phase with applications in coatings, adhesives, and purification membranes. Beyond macroscale phase separation, precision polymer synthesis has enabled further development of polyelectrolyte complex (PEC)-based self-assembled micelles and hydrogels with applications in biotechnology. Interestingly, it has been suggested that mechanisms similar to polyelectrolyte complexation drive formation of biological condensates that play an indispensable role in cellular biogenesis. The formation pathways and functionality of these complex materials is dependent on the physical properties that are built into polymer structure and the resulting physical conformation in the dilute and dense phase. Scattering techniques have enabled in situ investigation of structure-function relationships in PEC materials that may address unresolved biophysical questions in cellular processes as well as catalyze the development of novel materials for diverse applications. We describe preparation of PEC materials with controlled polymer characteristics (length, blockiness, charge density), small-angle X-ray scattering (SAXS) techniques employed to probe appropriate length scales, and the data analysis routines from a practical standpoint for new users. This article deals with bulk complexes and not with the related, important and interesting area of non-equilibrium layer-by-layer assembly of polyelectrolytes.

Impact of wet-dry cycling on the phase behavior and compartmentalization properties of complex coacervates.

Wet-dry cycling on the early Earth is thought to have facilitated production of molecular building blocks of life, but its impact on self-assembly and compartmentalization remains largely unexplored. Here, we investigate dehydration/rehydration of complex coacervates, which are membraneless compartments formed by phase separation of polyelectrolyte solutions. Solution compositions are identified for which tenfold water loss results in maintenance, disappearance, or appearance of coacervate droplets. Systems maintaining coacervates throughout the dehydration process are further evaluated to understand how their compartmentalization properties change with drying. Although added total RNA concentrations increase tenfold, RNA concentration within coacervates remains steady. Exterior RNA concentrations rise, and exchange rates for encapsulated versus free RNAs increase with dehydration. We explain these results in light of the phase diagram, with dehydration-driven ionic strength increase being particularly important in determining coacervate properties. This work shows that wet-dry cycling can alter the phase behavior and protocell-relevant functions of complex coacervates.

Comparing Zwitterionic and PEG Exteriors of Polyelectrolyte Complex Micelles.

A series of model polyelectrolyte complex micelles (PCMs) was prepared to investigate the consequences of neutral and zwitterionic chemistries and distinct charged cores on the size and stability of nanocarriers. Using aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization, we synthesized a well-defined diblock polyelectrolyte system, poly(2-methacryloyloxyethyl phosphorylcholine methacrylate)-block-poly((vinylbenzyl) trimethylammonium) (PMPC-PVBTMA), at various neutral and charged block lengths to compare directly against PCM structure-property relationships centered on poly(ethylene glycol)-block-poly((vinylbenzyl) trimethylammonium) (PEG-PVBTMA) and poly(ethylene glycol)-block-poly(l-lysine) (PEG-PLK). After complexation with a common polyanion, poly(sodium acrylate), the resulting PCMs were characterized by dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). We observed uniform assemblies of spherical micelles with a diameter ~1.5-2× larger when PMPC-PVBTMA was used compared to PEG-PLK and PEG-PVBTMA via SAXS and DLS. In addition, PEG-PLK PCMs proved most resistant to dissolution by both monovalent and divalent salt, followed by PEG-PVBTMA then PMPC-PVBTMA. All micelle systems were serum stable in 100% fetal bovine serum over the course of 8 h by time-resolved DLS, demonstrating minimal interactions with serum proteins and potential as in vivo drug delivery vehicles. This thorough study of the synthesis, assembly, and characterization of zwitterionic polymers in PCMs advances the design space for charge-driven micelle assemblies.

Spatiotemporal Formation and Growth Kinetics of Polyelectrolyte Complex Micelles with Millisecond Resolution.

We have directly observed the in situ self-assembly kinetics of polyelectrolyte complex (PEC) micelles by synchrotron time-resolved small-angle X-ray scattering, equipped with a stopped-flow device that provides millisecond temporal resolution. A synthesized neutral-charged diblock polycation and homopolyanion that we have previously investigated as a model charge-matched, core-shell micelle system were selected for this work. The initial micellization of the oppositely charged polyelectrolytes was completed within the dead time of mixing of 100 ms, followed by micelle growth and equilibration up to several seconds. By combining the structural evolution of the radius of gyration (Rg) with complementary molecular dynamics simulations, we show how the self-assemblies evolve incrementally in size over time through a two-step kinetic process: first, oppositely charged polyelectrolyte chains pair to form nascent aggregates that immediately assemble into spherical micelles, and second, these PEC micelles grow into larger micellar entities. This work has determined one possible kinetic pathway for the initial formation of PEC micelles, which provides useful physical insights for increasing fundamental understanding self-assembly dynamics, driven by polyelectrolyte complexation that occurs on ultrafast time scales.

Evaluating Large-Scale STEM Outreach Efficacy with a Consistent Theme: Thermodynamics for Elementary School Students.

A biannual chemistry demonstration-based show named "Energy and U" was created to extend the general outreach themes of science, technology, engineering, and mathematics (STEM) fields and a college education with a specific goal: to teach the first law of thermodynamics to elementary school students. The effectiveness of the program was analyzed using a clicker survey system for over 12 000 visiting students. The fraction of the students that correctly answered the question "Is it possible to create energy?" increased from 14% immediately before the show to 89% immediately after the show. Students who had seen the show at least 5 months prior were twice as likely to correctly answer at the beginning of the show, demonstrating longer-term lesson retention. Interestingly, similar trends were observed for the adult chaperones that accompanied the students and participated in the clicker survey. A statistically significant difference (>99% confidence interval) was noted between the students' responses to the questions "Can you create energy?" and "Can you destroy energy?", revealing a potential effect of word choice on the interpretation of the first law of thermodynamics despite the two questions representing complementary concepts. Student performance, measured interest in science, and desire to attend college were not correlated with standard economic indicators. This measurement is consistent with the postulate that economic biases surrounding interest in STEM fields are less pronounced in elementary school than later in high school.

Polyelectrolyte Complexation of Oligonucleotides by Charged Hydrophobic-Neutral Hydrophilic Block Copolymers.

Polyelectrolyte complex micelles (PCMs, core-shell nanoparticles formed by complexation of a polyelectrolyte with a polyelectrolyte-hydrophilic neutral block copolymer) offer a solution to the critical problem of delivering therapeutic nucleic acids, Despite this, few systematic studies have been conducted on how parameters such as polycation charge density, hydrophobicity, and choice of charged group influence PCM properties, despite evidence that these strongly influence the complexation behavior of polyelectrolyte homopolymers. In this article, we report a comparison of oligonucleotide PCMs and polyelectrolyte complexes formed by poly(lysine) and poly((vinylbenzyl) trimethylammonium) (PVBTMA), a styrenic polycation with comparatively higher charge density, increased hydrophobicity, and a permanent positive charge. All of these differences have been individually suggested to provide increased complex stability, but we find that PVBTMA in fact complexes oligonucleotides more weakly than does poly(lysine), as measured by stability versus added salt. Using small angle X-ray scattering and electron microscopy, we find that PCMs formed from both cationic blocks exhibit very similar structure-property relationships, with PCM radius determined by the cationic block size and shape controlled by the hybridization state of the oligonucleotides. These observations narrow the design space for optimizing therapeutic PCMs and provide new insights into the rich polymer physics of polyelectrolyte self-assembly.

Interparticle Interactions in Dilute Solutions of Polyelectrolyte Complex Micelles.

The application of dilute solutions of polyelectrolyte complex (PEC) micelles for delivering therapeutic nucleic acids into disease sites has gained momentum. This Letter reports a detailed characterization of PEC micelles in dilute solutions including their internal structures and the determination of the interparticle interactions. The polymer concentration ranges from 0.1 to 0.5 wt %, a regime where micelle-micelle interactions are infrequent. We employ synchrotron small-angle X-ray scattering (SAXS) to simultaneously probe the morphology, internal structure, and radius of gyration (Rg) of the self-assemblies formed by charged diblock polyelectrolytes and homopolyelectrolytes. The emerging appearance of the structure factor in SAXS profiles with the increasing polymer concentration demonstrates the presence of the repulsive intermicellar correlations, which is further confirmed by the differences between the "reciprocal Rg" estimated by Guinier approximation and the "real space Rg" determined by pair distribution functions. We find that the soft corona chains tethered on the surface of phase-separated complex domains are compressed when micelles come close to the point where a hard-sphere interaction takes over. These findings contribute to the fundamental understanding of the structure and space-filling constraints in the complexation-driven self-assemblies and advance the rational design of cationic polymer-based nonviral gene delivery vectors.

Controlling Complex Coacervation via Random Polyelectrolyte Sequences.

The utilization of chemical sequence control in polymeric materials is key to enabling material design on par with biomacromolecular systems. One important avenue for scalable sequence-controlled polymers leverages the random copolymerization of distinct monomers, with the statistical distribution of the monomeric sequence arising from reaction kinetics following a first-order Markov process. Here we utilize the framework of the random phase approximation (RPA) to develop a theory for the phase behavior of symmetric polyelectrolyte coacervates whose chemical sequences are dictated by simple statistical distributions. We find that a high charge "blockiness" within the random sequences favors the formation of denser and more salt-resistant coacervates while simultaneously increasing the width of the two-phase region. We trace these physical effects to the increased cooperativity of Coulomb interactions that results from increased charge blockiness in oppositely charged polyelectrolytes.

Non-equilibrium phenomena and kinetic pathways in self-assembled polyelectrolyte complexes.

Polyelectrolyte complexation has been conventionally focused on the thermodynamic states, where assemblies have equilibrated in solutions. Far less attention has been given to complex systems that are kinetically trapped at non-equilibrium states. A combination of time-resolved dynamic light scattering, small angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (Cryo-TEM) was employed here to investigate the internal structures and morphological evolution of non-equilibrium aggregates forming from a pair of two strong block polyelectrolytes over wide time and length scales. The role of formation pathways of electrostatically driven aggregates was assessed using two processing protocols: direct dissolution and salt annealing. The former led to thermodynamically stable products, while the latter resulted in kinetically trapped transient structures. After adding salt, the metastable structures gradually transformed into stable products. Cryo-TEM images showed the interconnected irregular morphologies of the aggregates, and SAXS data revealed the presence of fuzzy globular complexes with R g ∼ 10 nm within them. A two-step process in the time-dependent structural transformation was found and characterized by a fast breakdown of interconnected transient aggregates followed by a slow redistribution of the incipient individual electrostatic assemblies. Furthermore, the prolonged aggregate disintegration process fitting to a stretched exponential function unveiled the broad relaxation distribution and significant structural heterogeneity in these polyelectrolyte complex nanoaggregates. This work brings new insight into the comprehension of non-equilibrium phenomena in self-assembled electrostatic assemblies and represents a first step toward constructing far-from-equilibrium polyelectrolyte complexes de novo for future applications.

Advances in Polymer Design for Enhancing Oral Drug Solubility and Delivery.

Synthetic polymers have enabled amorphous solid dispersions (ASDs) to emerge as an oral delivery strategy for overcoming poor drug solubility in aqueous environments. Modern ASD products noninvasively treat a range of chronic diseases (for example, hepatitis C, cystic fibrosis, and HIV). In such formulations, polymeric carriers generate and maintain drug supersaturation upon dissolution, increasing the apparent drug solubility to enhance gastrointestinal barrier absorption and oral bioavailability. In this Review, we outline several approaches in designing polymeric excipients to drive interactions with active pharmaceutical ingredients (APIs) in spray-dried ASDs, highlighting polymer-drug formulation guidelines from industrial and academic perspectives. Special attention is given to new commercial and specialized polymer design strategies that can solubilize highly hydrophobic APIs and suppress the propensity for rapid drug recrystallization. These molecularly customized excipients and hierarchical excipient assemblies are promising toward informing early-stage drug-discovery development and reformulating existing API candidates into potentially lifesaving oral medicines for our growing global population.

Open-to-Air RAFT Polymerization in Complex Solvents: From Whisky to Fermentation Broth.

We investigate the use of in situ enzyme degassing to facilitate the open-to-air reversible addition-fragmentation chain transfer (RAFT) polymerization of hydroxyethyl acrylate (HEA) in a wide range of complex aqueous solvents, including, beer, wine, liquor, and fermentation broth. This enzyme-assisted polymerization procedure is impressively robust, and poly(HEA) was attained with good control over molecular weight and a narrow dispersity in nearly all of the solvents tested. Kinetics experiments on HEA polymerization in whisky and spectroscopic analysis of the purified polymers suggest high end-group fidelity, as does the successful chain extension of a poly(HEA) macro chain transfer agent with narrow dispersity. These results suggest enzyme-assisted RAFT may be a powerful and underutilized tool for high-throughput screening and materials discovery and may simplify the synthesis of well-defined polymers in complex conditions.

Synthesis and Assembly of Designer Styrenic Diblock Polyelectrolytes.

Harnessing molecular design principles toward functional applications of ion-containing macromolecules relies on diversifying experimental data sets of well-understood materials. Here, we report a simple, tunable framework for preparing styrenic polyelectrolytes, using aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization in a parallel synthesis approach. A series of diblock polycations and polyanions were RAFT chain-extended from poly(ethylene oxide) (PEO) using (vinylbenzyl)trimethylammonium chloride (PEO-b-PVBTMA) and sodium 4-styrenesulfonate (PEO-b-PSS), with varying neutral PEO block lengths, charged styrenic block lengths, and RAFT end-group identity. The materials characterization and kinetics study of chain growth exhibited control of the molar mass distribution for both systems. These block polyelectrolytes were also demonstrated to form polyelectrolyte complex (PEC) driven self-assemblies. We present two simple outcomes of micellization to show the importance of polymer selection from a broadened pool of polyelectrolyte candidates: (i) uniform PEC-core micelles comprising PEO-b-PVBTMA and poly(acrylic acid) and (ii) PEC nanoaggregates comprising PEO-b-PVBTMA and PEO-b-PSS. The materials characteristics of these charged assemblies were investigated with dynamic light scattering, small-angle X-ray scattering, and cryogenic-transmission electron microscopy imaging. This model synthetic platform offers a straightforward path to expand the design space of conventional polyelectrolytes into gram-scale block polymer structures, which can ultimately enable the development of more sophisticated ionic materials into technology.

Molecular engineering solutions for therapeutic peptide delivery.

Proteins and their interactions in and out of cells must be well-orchestrated for the healthy functioning and regulation of the body. Even the slightest disharmony can cause diseases. Therapeutic peptides are short amino acid sequences (generally considered <50 amino acids) that can naturally mimic the binding interfaces between proteins and thus, influence protein-protein interactions. Because of their fidelity of binding, peptides are a promising next generation of personalized medicines to reinstate biological harmony. Peptides as a group are highly selective, relatively safe, and biocompatible. However, they are also vulnerable to many in vivo pharmacologic barriers limiting their clinical translation. Current advances in molecular, chemical, and nanoparticle engineering are helping to overcome these previously insurmountable obstacles and improve the future of peptides as active and highly selective therapeutics. In this review, we focus on self-assembled vehicles as nanoparticles to carry and protect therapeutic peptides through this journey, and deliver them to the desired tissue.

High-Throughput Excipient Discovery Enables Oral Delivery of Poorly Soluble Pharmaceuticals.

Polymeric excipients are crucial ingredients in modern pills, increasing the therapeutic bioavailability, safety, stability, and accessibility of lifesaving products to combat diseases in developed and developing countries worldwide. Because many early-pipeline drugs are clinically intractable due to hydrophobicity and crystallinity, new solubilizing excipients can reposition successful and even failed compounds to more effective and inexpensive oral formulations. With assistance from high-throughput controlled polymerization and screening tools, we employed a strategic, molecular evolution approach to systematically modulate designer excipients based on the cyclic imide chemical groups of an important (yet relatively insoluble) drug phenytoin. In these acrylamide- and methacrylate-containing polymers, a synthon approach was employed: one monomer served as a precipitation inhibitor for phenytoin recrystallization, while the comonomer provided hydrophilicity. Systems that maintained drug supersaturation in amorphous solid dispersions were identified with molecular-level understanding of noncovalent interactions using NOESY and DOSY NMR spectroscopy. Poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (poly(NIPAm-co-DMA)) at 70 mol % NIPAm exhibited the highest drug solubilization, in which phenytoin associated with inhibiting NIPAm units only with lowered diffusivity in solution. In vitro dissolution tests of select spray-dried dispersions corroborated the screening trends between polymer chemical composition and solubilization performance, where the best NIPAm/DMA polymer elevated the mean area-under-the-dissolution-curve by 21 times its crystalline state at 10 wt % drug loading. When administered to rats for pharmacokinetic evaluation, the same leading poly(NIPAm-co-DMA) formulation tripled the oral bioavailability compared to a leading commercial excipient, HPMCAS, and translated to a remarkable 23-fold improvement over crystalline phenytoin.

The modeling of medical expenditure data from a longitudinal survey using the generalized method of moments (GMM) approach.

Medical expenditure data analysis has recently become an important problem in biostatistics. These data typically have a number of features making their analysis rather difficult. Commonly, they are heavily right-skewed, contain a large percentage of zeros, and often exhibit large numbers of missing observations because of death and/or the lack of follow-up. They are also commonly obtained from records that are linked to large longitudinal data surveys. In this manuscript, we suggest a novel approach to modeling these data through the use of generalized method of moments estimation procedure combined with appropriate weights that account for both dropout due to death and the probability of being sampled from among the National Long Term Care Survey (NLTCS) subjects. This approach seems particularly appropriate because of the large number of subjects relative to the length of observation period (in months). We also use a simulation study to compare our proposed approach with and without the use of weights. The proposed model is applied to medical expenditure data obtained from the 2004-2005 NLTCS-linked Medicare database. The results suggest that the amount of medical expenditures incurred is strongly associated with higher number of activities of daily living (ADL) disabilities and self-reports of unmet need for help with ADL disabilities. Copyright © 2016 John Wiley & Sons, Ltd.