PEGylation for enhancing nanoparticle diffusion in mucus

The viscoelastic mucus secretions coating exposed organs such as the lung airways and the female reproductive tract can trap and quickly eliminate not only foreign pathogens and ultrafine particles but also particle-based drug delivery systems, thus limiting sustained and targeted drug delivery at mucosal surfaces. To improve particle distribution across the mucosa and enhance delivery to the underlying epithelium, many investigators have sought to develop nanoparticles capable of readily traversing mucus. The first synthetic nanoparticles shown capable of rapidly penetrating physiological mucus secretions utilized a dense coating of polyethylene glycol (PEG) covalently grafted onto the surface of preformed polymeric nanoparticles. In the decade since, PEG has become the gold standard in engineering mucus-penetrating drug carriers for sustained and targeted drug delivery to the lungs, gastrointestinal tract, eyes, and female reproductive tract. This review summarizes the history of the development of various PEG-based mucus-penetrating particles, and highlights the key physicochemical properties of PEG coatings and PEGylation strategies to achieve muco-inert PEG coatings on nanoparticle drug carriers for improved drug and gene delivery at mucosal surfaces

Limited processivity of single motors improves overall transport flux of self-assembled motor-cargo complexes

Single kinesin molecular motors can processively move along a microtubule (MT) a few micrometers on average before dissociating. However, cellular length scales over which transport occurs are several hundred microns and more. Why seemingly unreliable motors are used to transport cellular cargo remains poorly understood. We propose a new theory for how low processivity, the average length of a single bout of directed motion, can enhance cellular transport when motors and cargoes must first diffusively self assemble into complexes. We employ stochastic modeling to determine the effect of processivity on overall cargo transport flux. We show that, under a wide range of physiologically relevant conditions, possessing "infinite" processivity does not maximize flux along MTs. Rather, we find that low processivity i.e., weak binding of motors to MTs, is optimal. These results shed light on the relationship between processivity and transport efficiency and offer a new theory for the physiological benefits of low motor processivity

Minimizing biases associated with tracking analysis of submicron particles in heterogeneous biological fluids

Tracking the dynamic motion of individual nanoparticles or viruses offers quantitative insights into their real-time behavior and fate in different biological environments. Indeed, particle tracking is a powerful tool that has facilitated the development of drug carriers with enhanced penetration of mucus, brain tissues and other extracellular matrices. Nevertheless, heterogeneity is a hallmark of nanoparticle diffusion in such complex environments: identical particles can exhibit strongly hindered or unobstructed diffusion within microns of each other. The common practice in 2D particle tracking, namely analyzing all trackable particle traces with equal weighting, naturally biases toward rapidly diffusing sub-populations at shorter time scales. This in turn results in misrepresentation of particle behavior and a systematic underestimate of the time necessary for a population of nanoparticles to diffuse specific distances. We show here via both computational simulation and experimental data that this bias can be rigorously corrected by weighing the contribution by each particle trace on a ‘frame-by-frame’ basis. We believe this methodology presents an important step towards objective and accurate assessment of the heterogeneous transport behavior of submicron drug carriers and pathogens in biological environments

Enhanced Trapping of HIV-1 by Human Cervicovaginal Mucus Is Associated with Lactobacillus crispatus -Dominant Microbiota

ABSTRACT Cervicovaginal mucus (CVM) can provide a barrier that precludes HIV and other sexually transmitted virions from reaching target cells in the vaginal epithelium, thereby preventing or reducing infections. However, the barrier properties of CVM differ from woman to woman, and the causes of these variations are not yet well understood. Using high-resolution particle tracking of fluorescent HIV-1 pseudoviruses, we found that neither pH nor Nugent scores nor total lactic acid levels correlated significantly with virus trapping in unmodified CVM from diverse donors. Surprisingly, HIV-1 was generally trapped in CVM with relatively high concentrations of d -lactic acid and a Lactobacillus crispatus -dominant microbiota. In contrast, a substantial fraction of HIV-1 virions diffused rapidly through CVM with low concentrations of d -lactic acid that had a Lactobacillus iners -dominant microbiota or significant amounts of Gardnerella vaginalis , a bacterium associated with bacterial vaginosis. Our results demonstrate that the vaginal microbiota, including specific species of Lactobacillus , can alter the diffusional barrier properties of CVM against HIV and likely other sexually transmitted viruses and that these microbiota-associated changes may account in part for the elevated risks of HIV acquisition linked to bacterial vaginosis or intermediate vaginal microbiota. IMPORTANCE Variations in the vaginal microbiota, especially shifts away from Lactobacillus -dominant microbiota, are associated with differential risks of acquiring HIV or other sexually transmitted infections. However, emerging evidence suggests that Lactobacillus iners frequently colonizes women with recurring bacterial vaginosis, raising the possibility that L. iners may not be as protective as other Lactobacillus species. Our study was designed to improve understanding of how the cervicovaginal mucus barrier against HIV may vary between women along with the vaginal microbiota and led to the finding that the vaginal microbiota, including specific species of Lactobacillus , can directly alter the diffusional barrier properties of cervicovaginal mucus. This work advances our understanding of the complex barrier properties of mucus and highlights the differential protective ability of different species of Lactobacillus , with Lactobacillus crispatus and possibly other species playing a key role in protection against HIV and other sexually transmitted infections. These findings could lead to the development of novel strategies to protect women against HIV

Anti-PEG immunity: emergence, characteristics, and unaddressed questions: Anti-PEG immunity

The modification of protein and nanoparticle therapeutics with polyethylene glycol (PEG), a flexible, uncharged and highly hydrophilic polymer, is a widely adopted approach to reduce RES clearance, extend circulation time, and improve drug efficacy. Nevertheless, an emerging body of literature, generated by numerous research groups, demonstrates that the immune system can produce antibodies that specifically bind PEG, which can lead to the “accelerated blood clearance” of PEGylated therapeutics. In animals, anti-PEG immunity is typically robust but short-lived and consists of a predominantly anti-PEG IgM response. Rodent studies suggest that the induction of anti-PEG antibodies (α-PEG Abs) primarily occurs through a type 2 T-cell independent mechanism. Although anti-PEG immunity is less well-studied in humans, the presence of α-PEG Abs has been correlated with reduced efficacy of PEGylated therapeutics in clinical trials. The prevalence of anti-PEG IgG and reports of memory immune responses, as well as the existence of α-PEG Abs in healthy untreated individuals, suggests that the mechanism(s) and features of human anti-PEG immune responses may differ from those of animal models. Many questions, including the incidence rate of pre-existing α-PEG Abs and immunological mechanism(s) of α-PEG Ab formation in humans, must be answered in order to fully address the potential complications of anti-PEG immunity

Challenges & opportunities for phage-based in situ microbiome engineering in the gut

The gut microbiome is a promising target for the development of GI tract therapies, yet it has been under-exploited due, in part, to a lack of tools to control and manipulate complex microbial communities. To date, the most common approach in harnessing bacteria for therapeutic purposes has been to deliver ex vivo engineered bacteria-effectively taking a bacterial cell therapy-based approach. An alternative approach involves taking advantage of the rich microbial ecosystem in the gut by genetically modifying the microbiome in situ through the use of engineered bacteriophages-akin to human gene therapies delivered by viral vectors. In this review, we present the challenges and opportunities associated with engineering bacteriophages to control and manipulate the gut microbiome

Siglec15/TGF‐β bispecific antibody mediates synergistic anti‐tumor response against 4T1 triple negative breast cancer in mice

Abstract An ideal tumor‐specific immunomodulatory therapy should both preferentially target the tumor, while simultaneously reduce the immunosuppressive environment within the tumor. This guiding principle led us to explore engineering Siglec‐15 (S15) targeted bispecific antibody (bsAb) to enhance therapy against triple negative breast cancer (TNBC). S15 appears to be exclusively expressed on macrophages and diverse tumor cells, including human and mouse 4T1 TNBC. TGF‐β is a growth hormone frequently associated with increased tumor invasiveness, including in TNBC. Here, to overcome the immune‐suppressive environment within TNBC tumors to enable more effective cancer therapy, we engineered a bispecific antibody (bsAb) targeting both Siglec15 and TGF‐β. In mice engrafted with orthotopic 4T1 tumors, S15/TGF‐β bsAb treatment was highly effective in suppressing tumor growth, not only compared to control monoclonal antibody (mAb) but also markedly more effective than mAbs against S15 alone, against TGF‐β alone, as well as a cocktail of both anti‐S15 and anti‐TGF‐β mAbs. We did not detect liver and lung metastasis in mice treated with S15/TGF‐β bsAb, unlike all other treatment groups at the end of the study. The enhanced anti‐tumor response observed with S15/TGF‐β bsAb correlated with a less immunosuppressive environment in the tumor. These results underscore S15‐targeted bsAb as a promising therapeutic strategy for TNBC, and possibly other S15 positive solid tumors

Experimental Data and PBPK Modeling Quantify Antibody Interference in PEGylated Drug Carrier Delivery

Physiologically-based pharmacokinetic (PBPK) modeling is a popular drug development tool that integrates physiology, drug physicochemical properties, preclinical data, and clinical information to predict drug systemic disposition. Since PBPK models seek to capture complex physiology, parameter uncertainty and variability is a prevailing challenge: there are often more compartments (e.g., organs, each with drug flux and retention mechanisms, and associated model parameters) than can be simultaneously measured. To improve the fidelity of PBPK modeling, one approach is to search and optimize within the high-dimensional model parameter space, based on experimental time-series measurements of drug distributions. Here, we employ Latin Hypercube Sampling (LHS) on a PBPK model of PEG-liposomes (PL) that tracks biodistribution in an 8-compartment mouse circulatory system, in the presence (APA+) or absence (naive) of anti-PEG antibodies (APA). Near-continuous experimental measurements of PL concentration during the first hour post-injection from the liver, spleen, kidney, muscle, lung, and blood plasma, based on PET/CT imaging in live mice, are used as truth sets with LHS to infer optimal parameter ranges for the full PBPK model. The data and model quantify that PL retention in the liver is the primary differentiator of biodistribution patterns in naive versus APA+ mice, and spleen the secondary differentiator. Retention of PEGylated nanomedicines is substantially amplified in APA+ mice, likely due to PL-bound APA engaging specific receptors in the liver and spleen that bind antibody Fc domains. Our work illustrates how applying LHS to PBPK models can further mechanistic understanding of the biodistribution and antibody-mediated clearance of specific drugs