Evaluating the oral delivery of GalNAc-conjugated siRNAs in rodents and non-human primates.

Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution.

Modulation of carcinogenesis with selected GRAS nutraceuticals via Keap1-Nrf2 signaling pathway.

Keap1-Nrf2 is a fundamental signaling cascade known to promote or prevent carcinogenesis. Extensive studies identify the key target of modulatory aspects of Keap1-Nrf2 signaling against cancer. Nutraceuticals are those dietary agents with many health benefits that have immense potential for cancer chemoprevention. The nutritional supplements known as nutraceuticals are found to be one of the most promising chemoprevention agents. Upon investigating the dual nature of Nrf2, it became clear that, in addition to shielding normal cells from numerous stresses, Nrf2 may also promote the growth of tumors. In the present review, we performed a systematic analysis of the role of 12 different nutraceuticals like curcumin, sulforaphane, resveratrol, polyunsaturated fatty acids (PUFA) from fish oil, lycopene, soybean, kaempferol, allicin, thymoquinone, quercetin, gingerol, and piperine in modulating the Nrf2/Keap1 signaling mechanism. Among these, 12 Generally Recognized As Safe (GRAS) certified nutraceuticals, sulforaphane is the most extensively studied compound in modulating Keap1-Nrf signaling. Even though there is much evidence at preclinical levels, further high-quality research is still required to validate the potential role of these nutraceuticals in Keap1-Nrf2 modulation.

The Nonclinical Disposition and Pharmacokinetic/Pharmacodynamic Properties of N-Acetylgalactosamine-Conjugated Small Interfering RNA Are Highly Predictable and Build Confidence in Translation to Human.

Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to N-acetylgalactosamine (GalNAc) ligands has become the primary strategy for hepatocyte-targeted delivery, and with the recent approvals of GIVLAARI (givosiran) for the treatment of acute hepatic porphyria, OXLUMO (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio (inclisiran) for the treatment of hypercholesterolemia, the technology has been well validated clinically. Although much knowledge has been gained over decades of development, there is a paucity of published literature on the drug metabolism and pharmacokinetic properties of GalNAc-siRNA. With this in mind, the goals of this minireview are to provide an aggregate analysis of these nonclinical absorption, distribution, metabolism, and excretion (ADME) data to build confidence on the translation of these properties to human. Upon subcutaneous administration, GalNAc-conjugated siRNAs are quickly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination through asialoglycoprotein receptor-mediated uptake from circulation into hepatocytes. These studies confirm that liver PK, including half-life and, most importantly, siRNA levels in RNA-induced silencing complex in hepatocytes, are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the ADME properties of GalNAc-conjugated siRNAs. These studies demonstrate that the PK/PD and ADME properties of GalNAc-conjugated siRNAs are highly conserved across species, are largely predictable, and can be accurately scaled to human, allowing us to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles. SIGNIFICANCE STATEMENT: Several nonclinical ADME studies have been conducted in order to provide a comprehensive overview of the disposition and elimination of GalNAc-conjugated siRNAs and the pharmacokinetic/pharmacodynamic translation between species. These studies demonstrate that the ADME properties of GalNAc-conjugated siRNAs are well correlated and predictable across species, building confidence in the ability to extrapolate to human.

Generation and validation of structurally defined antibody-siRNA conjugates.

Gene silencing by RNA interference (RNAi) has emerged as a powerful treatment strategy across a potentially broad range of diseases. Tailoring siRNAs to silence genes vital for cancer cell growth and function could be an effective treatment, but there are several challenges which must be overcome to enable their use as a therapeutic modality, among which efficient and selective delivery to cancer cells remains paramount. Attempts to use antibodies for siRNA delivery have been reported but these strategies use either nonspecific conjugation resulting in mixtures, or site-specific methods that require multiple steps, introduction of mutations, or use of enzymes. Here, we report a method to generate antibody-siRNA (1:2) conjugates (ARCs) that are structurally defined and easy to assemble. This ARC platform is based on engineered dual variable domain (DVD) antibodies containing a natural uniquely reactive lysine residue for site-specific conjugation to ß-lactam linker-functionalized siRNA. The conjugation is efficient, does not compromise the affinity of the parental antibody, and utilizes chemically stabilized siRNA. For proof-of-concept, we generated DVD-ARCs targeting various cell surface antigens on multiple myeloma cells for the selective delivery of siRNA targeting ß-catenin (CTNNB1). A set of BCMA-targeting DVD-ARCs at concentrations as low as 10 nM revealed significant CTNNB1 mRNA and protein knockdown.

Investigating the pharmacodynamic durability of GalNAc-siRNA conjugates.

One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc-siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc-siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.

Non-inertial lift induced migration for label-free sorting of cells in a co-flowing aqueous two-phase system.

Label free sorting of circulating tumor cells (CTCs) often remains a challenge due to their rarity in peripheral blood and identical morphology to white blood cells. We present a novel label-free passive microfluidic technique for isolation of cancer cells (EpCAM+ and CD45-) from peripheral blood mononuclear cells (PBMCs) (CD45+ and EpCAM-) in aqueous two-phase system (ATPS). Our technique involves non-inertial lift induced lateral cell migration across liquid-liquid interface that is employed for sorting cells of different size and stiffness. The interplay between lift force and interfacial tension (IFT) force governs cell migration phenomena. We estimate the order of magnitude of the lift force and find it to be higher than the IFT for cancer cells above a critical strain rate parameter ([small gamma, Greek, dot above]/h). The effect of spreading parameter and viscoelastic force was found to have negligible effect on lateral migration of cells. We demonstrated isolation of two different types of cancer cells, namely, MCF-7 and MDA-MB-231 from PBMCs and quantify our sorting results by tagging the cells with EpCAM and CD45 and using fluorescence imaging. With 102-104 cancer cells in 105-107 PBMCs, we achieved a processing rate of >25 000 cells per min at a sorting efficiency of ∼99%. Moreover, we demonstrated that cancer cells isolated from PBMCs using the proposed technique remain viable and can be cultured for downstream analysis.

Evaluation of GalNAc-siRNA Conjugate Activity in Pre-clinical Animal Models with Reduced Asialoglycoprotein Receptor Expression.

The hepatocyte-specific asialoglycoprotein receptor (ASGPR) is an ideal candidate for targeted drug delivery to the liver due to its high capacity for substrate clearance from circulation together with its well-conserved expression and function across species. The development of GalNAc-siRNA conjugates, in which a synthetic triantennary N-acetylgalactosamine-based ligand is conjugated to chemically modified siRNA, has enabled efficient, ASGPR-mediated delivery to hepatocytes. To investigate the potential impact of variations in receptor expression on the efficiency of GalNAc-siRNA conjugate delivery, we evaluated the pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates in multiple pre-clinical models with reduced receptor expression. Despite greater than 50% reduction in ASGPR levels, GalNAc conjugate activity was retained, suggesting that the remaining receptor capacity was sufficient to mediate efficient uptake of potent GalNAc-siRNAs at pharmacologically relevant dose levels. Collectively, our data support a broad application of the GalNAc-siRNA technology for hepatic targeting, including disease states where ASGPR expression may be reduced.

Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates.

Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5'-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5'-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc-siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates.

Droplet Demulsification Using Ultralow Voltage-Based Electrocoalescence.

Demulsification of droplets stabilized with surfactant is very challenging due to their low surface energy. We report ultralow voltage-based electrocoalescence phenomenon for the demulsification of aqueous droplets with an aqueous stream. In the absence of electric field, due to the disjoining pressure resulting from the tail-tail interaction between the surfactant molecules present on the aqueous droplets and interface, coalescence of aqueous droplets with the aqueous stream is prevented. However, above a critical electric field, the electrical stress overcomes the disjoining pressure, thus leading to the droplet coalescence. The influence of surfactant concentration, droplet diameter, and velocity on the electrocoalescence phenomena is studied. The macroscopic contact between the aqueous droplet with the aqueous stream enables droplet coalescence at much lower voltage (10 to 90 V), which is at least two orders of magnitude smaller than voltages used in prior works (1.0 to 3.0 kV). The electrocoalescence phenomena is used for the extraction of microparticles encapsulated in aqueous droplets into the aqueous stream and size-based selective demulsification. A new paradigm of droplet electrocoalescence and content extraction is presented that would find significant applications in chemistry and biology.

Self-Transport and Manipulation of Aqueous Droplets on Oil-Submerged Diverging Groove.

We report experimental study of self-transport of aqueous droplets along an oil-submerged diverging groove structure. The migration phenomenon is illustrated, and the effect of various parameters such as droplet size d, oil layer thickness h, groove angle 2θ, and groove thickness δ on the droplet transport behavior (i.e., migration velocity and length) is investigated. Our study reveals that complete engulfment of aqueous droplets in the oil layer, that is attributed to a positive spreading parameter ( S > 0), is a prerequisite for the droplet transport. The results show that only droplets of diameter larger than the oil layer thickness (i.e., d ≥ h) get transported owing to a differential Laplace pressure between the leading and trailing faces of a droplet because of the diverging groove. Using experimental data, the variation of droplet migration velocity with distance along the diverging groove is correlated as U( x) = ψ x-0.9, where ψ = d0.32θ-2.2 h-1.5δ0.7. The submerged groove structure was used to demonstrate simultaneous and sequential coalescence and transport of multiple droplets. Finally, the submerged groove structure was employed for extraction of aqueous droplets from oil. The proposed technique opens up a new avenue for evaporation and contamination free transport and coalescence of droplets for chemical and biological applications.

Continuous splitting of aqueous droplets at the interface of co-flowing immiscible oil streams in a microchannel.

We report the continuous splitting of aqueous droplets at the interface between two co-flowing immiscible oil streams in a microchannel. The aqueous droplets initially present in a primary continuous stream (CP1) migrate into a secondary continuous stream (CP2) when the ratio of the non-inertial lift force to the interfacial tension force exceeds a critical value (K. S. Jayaprakash, U. Banerjee and A. K. Sen, Langmuir, 2016, 32, 2136-2143). Here, experiments were performed to understand the droplet splitting phenomenon and demonstrate the splitting of droplets encapsulating microbeads and cells. The results showed that the droplet splitting phenomenon is governed by the capillary number Ca, which is a function of the average shear stress across the channel, interfacial tension σ between the CP1 and the droplet phase and the droplet length-scale L. Irrespective of the individual values of these parameters, droplet splitting was observed when the capillary number Ca exceeds a critical value Cacr, which was found to be a function of droplet to CP2 viscosity ratio λ. The Cacr was found to be minimum for λ ≈ 1 but higher for droplets of λ ≫ 1 and λ ≪ 1. The sizes of the primary and secondary daughter and migrated droplets (i.e. Lp|sD and Lp|sM) were found to increase linearly with the increase in the size of the primary or secondary parent droplets (Lp|sP). Splitting of parent droplets encapsulating a single microbead or PBMC showed that after splitting, the presence of the microbead or PBMC in the daughter or migrated droplets depends on the ratio of the size of the migrated droplets to that of the parent droplet (i.e. VM/VP). Finally, splitting of parent droplets containing two or more microbeads or cells into droplets containing a single particle or cell was demonstrated. A new paradigm of droplet splitting is reported that could find applications in soft matter and single-cell studies.

Dynamics of Aqueous Droplets at the Interface of Coflowing Immiscible Oils in a Microchannel.

We report the dynamics of aqueous droplets of different size and viscosity at the interface of a coflowing stream of immiscible oils (i.e., primary and secondary continuous phases) in a microchannel, at low Re. The lateral migration of droplets introduced into the primary continuous phase toward the interface and subsequent selective migration of droplets across the interface into the secondary continuous phase is investigated. The interplay between the competing noninertial lift and interfacial tension forces, which govern the interfacial migration of the droplets, is presented and discussed. The velocity and strain rate profiles, and interface location, which are critical for calculating the lift force and migration behavior of droplets, are presented. The trajectories of droplets of different size and viscosity in the primary continuous phase are obtained for different interface locations. During interfacial migration, the deformation behavior of droplets of different viscosities is studied. Finally, sorting of droplets based on size contrast is demonstrated and sorting efficiency is found. A new paradigm of migration and sorting of droplets is reported, which could find importance in chemical and biological applications.

Consecutive transitions from localized to delocalized transport states in the anharmonic chain of three coupled oscillators.

In the present paper, we study the mechanism of formation and bifurcations of highly nonstationary regimes manifested by different energy transport intensities, emerging in an anharmonic trimer model. The basic model under investigation comprises a chain of three coupled anharmonic oscillators subject to localized excitation, where the initial energy is imparted to the first oscillator only. We report the formation of three basic nonstationary transport states traversed by locally excited regimes. These states differ by spatial energy distribution, as well as by the intensity of energy transport along the chain. In the current study, we focus on numerical and analytical investigation of the intricate resonant mechanism governing the inter-state transitions of locally excited regimes. Results of the analytical study are in good agreement with the numerical simulations of the trimer model.

Sequence-Defined Oligomers from Hydroxyproline Building Blocks for Parallel Synthesis Applications.

The functionality of natural biopolymers has inspired significant effort to develop sequence-defined synthetic polymers for applications including molecular recognition, self-assembly, and catalysis. Conjugation of synthetic materials to biomacromolecules has played an increasingly important role in drug delivery and biomaterials. We developed a controlled synthesis of novel oligomers from hydroxyproline-based building blocks and conjugated these materials to siRNA. Hydroxyproline-based monomers enable the incorporation of broad structural diversity into defined polymer chains. Using a perfluorocarbon purification handle, we were able to purify diverse oligomers through a single solid-phase extraction method. The efficiency of synthesis was demonstrated by building 14 unique trimers and 4 hexamers from 6 diverse building blocks. We then adapted this method to the parallel synthesis of hundreds of materials in 96-well plates. This strategy provides a platform for the screening of libraries of modified biomolecules.

Hepatocyte-specific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo.

We recently demonstrated that siRNAs conjugated to triantennary N-acetylgalactosamine (GalNAc) induce robust RNAi-mediated gene silencing in the liver, owing to uptake mediated by the asialoglycoprotein receptor (ASGPR). Novel monovalent GalNAc units, based on a non-nucleosidic linker, were developed to yield simplified trivalent GalNAc-conjugated oligonucleotides under solid-phase synthesis conditions. Synthesis of oligonucleotide conjugates using monovalent GalNAc building blocks required fewer synthetic steps compared to the previously optimized triantennary GalNAc construct. The redesigned trivalent GalNAc ligand maintained optimal valency, spatial orientation, and distance between the sugar moieties for proper recognition by ASGPR. siRNA conjugates were synthesized by sequential covalent attachment of the trivalent GalNAc to the 3'-end of the sense strand and resulted in a conjugate with in vitro and in vivo potency similar to that of the parent trivalent GalNAc conjugate design.

Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing.

Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.

Single-Fraction Stereotactic Ablative Body Radiotherapy for Primary and Extracranial Oligometastatic Cancers.

Stereotactic ablative body radiotherapy (SABR) consists of delivering high doses of ionising radiation, typically across three to eight fractions with high precision and conformity. SABR has become increasingly commonplace throughout the last quarter of a century and is offered for the treatment of various primary and metastatic tumour types. Delivering SABR in a single fraction has arisen as an appealing possibility for several reasons. These include fewer hospital visits, greater patient convenience, improved sustainability and lower costs. However, these factors must be balanced against considerations such as toxicity, side-effects and, most importantly, progression-free and overall survival. In this review we seek to analyse the results of studies looking at the efficacy of single-fraction SABR for lung, prostate, renal and pancreas primary tumours, as well as oligometastases. The tumour type to be most widely treated with single-fraction SABR is lung, but its remit continues to expand. We also look at the biological rationale underpinning SABR and how this can be extended to single-fraction regimens. Finally, we turn our attention towards the future directions of SABR and specifically single-fraction regimens. These include the possibility of combining SABR with immunotherapy and technological advances in the field, which could serve to expand the scope of SABR. We conclude by summarising the current clinical studies of single-fraction SABR.

Impact of the COVID-19 Pandemic on Outcomes for Patients with Lung Cancer Receiving Curative-intent Radiotherapy in the UK.

AIMS: Previous work found that during the first wave of the COVID-19 pandemic, 34% of patients with lung cancer treated with curative-intent radiotherapy in the UK had a change to their centre's usual standard of care treatment (Banfill et al. Clin Oncol 2022;34:19-27). We present the impact of these changes on patient outcomes. MATERIALS AND METHODS: The COVID-RT Lung database was a prospective multicentre UK cohort study including patients with stage I-III lung cancer referred for and/or treated with radical radiotherapy between April and October 2020. Data were collected on patient demographics, radiotherapy and systemic treatments, toxicity, relapse and death. Multivariable Cox and logistic regression were used to assess the impact of having a change to radiotherapy on survival, distant relapse and grade ≥3 acute toxicity. The impact of omitting chemotherapy on survival and relapse was assessed using multivariable Cox regression. RESULTS: Patient and follow-up forms were available for 1280 patients. Seven hundred and sixty-five (59.8%) patients were aged over 70 years and 603 (47.1%) were female. The median follow-up was 213 days (119, 376). Patients with stage I-II non-small cell lung cancer (NSCLC) who had a change to their radiotherapy had no significant increase in distant relapse (P = 0.859) or death (P = 0.884); however, they did have increased odds of grade ≥3 acute toxicity (P = 0.0348). Patients with stage III NSCLC who had a change to their radiotherapy had no significant increase in distant relapse (P = 0.216) or death (P = 0.789); however, they did have increased odds of grade ≥3 acute toxicity (P < 0.001). Patients with stage III NSCLC who had their chemotherapy omitted had no significant increase in distant relapse (P = 0.0827) or death (P = 0.0661). CONCLUSION: This study suggests that changes to radiotherapy and chemotherapy made in response to the COVID-19 pandemic did not significantly affect distant relapse or survival. Changes to radiotherapy, namely increased hypofractionation, led to increased odds of grade ≥3 acute toxicity. These results are important, as hypofractionated treatments can help to reduce hospital attendances in the context of potential future emergency situations.

Metabolically Stable Anomeric Linkages Containing GalNAc-siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathways.

Conjugation of synthetic triantennary N-acetyl-d-galactosamine (GalNAc) to small interfering RNA (siRNA) mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake and siRNA-mediated gene silencing. The natural ß-glycosidic bond of the GalNAc ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands with S-, and C-glycosides with both α- and ß-anomeric linkages, N-glycosides with ß-anomeric linkage, and the O-glycoside with α-anomeric linkage were synthesized and conjugated to siRNA either on-column during siRNA synthesis or through a high-throughput, post-synthetic method. Unlike natural GalNAc, modified ligands were resistant to glycosidase activity. The siRNAs conjugated to newly designed ligands had similar affinities for ASGPR and similar silencing activity in mice as the parent GalNAc-siRNA conjugate. These data suggest that other factors, such as protein-nucleic acid interactions and loading of the antisense strand into the RNA-induced silencing complex (RISC), are more critical to the duration of action than the stereochemistry and stability of the anomeric linkage between the GalNAc moiety of the ligand conjugated to the sense strand of the siRNA.

Branched multifunctional polyether polyketals: variation of ketal group structure enables unprecedented control over polymer degradation in solution and within cells.

Multifunctional biocompatible and biodegradable nanomaterials incorporating specific degradable linkages that respond to various stimuli and with defined degradation profiles are critical to the advancement of targeted nanomedicine. Herein we report, for the first time, a new class of multifunctional dendritic polyether polyketals containing different ketal linkages in their backbone that exhibit unprecedented control over degradation in solution and within the cells. High-molecular-weight and highly compact poly(ketal hydroxyethers) (PKHEs) were synthesized from newly designed α-epoxy-ω-hydroxyl-functionalized AB(2)-type ketal monomers carrying structurally different ketal groups (both cyclic and acyclic) with good control over polymer properties by anionic ring-opening multibranching polymerization. Polymer functionalization with multiple azide and amine groups was achieved without degradation of the ketal group. The polymer degradation was controlled primarily by the differences in the structure and torsional strain of the substituted ketal groups in the main chain, while for polymers with linear (acyclic) ketal groups, the hydrophobicity of the polymer may play an additional role. This was supported by the log P values of the monomers and the hydrophobicity of the polymers determined by fluorescence spectroscopy using pyrene as the probe. A range of hydrolysis half-lives of the polymers at mild acidic pH values was achieved, from a few minutes to a few hundred days, directly correlating with the differences in ketal group structures. Confocal microscopy analyses demonstrated similar degradation profiles for PKHEs within live cells, as seen in solution and the delivery of fluorescent marker to the cytosol. The cell viability measured by MTS assay and blood compatibility determined by complement activation, platelet activation, and coagulation assays demonstrate that PKHEs and their degradation products are highly biocompatible. Taken together, these data demonstrate the utility this new class of biodegradable polymer as a highly promising candidate in the development of multifunctional nanomedicine.