Polymers for extended-release administration.

Developing strategies to deliver the required dose of therapeutics into target tissues and cell populations within the body is a principal aim of controlled release and drug delivery. Specifically, there is an interest in developing formulations that can achieve drug concentrations within the therapeutic window, for extended periods of time, with tunable release profiles, and with minimal complication and distress for the patient. To date, drug delivery systems have been developed to serve as depots, triggers, and carriers for therapeutics including small molecules, biologics, and cell-based therapies. Notably, the efficacy of these systems is intricately tied to the manner in which they are administered. For example, systemic and oral routes of administration are common, but both can result in rapid clearance from the organism. Towards this end, what formulation and administration route strategies are available to prolong the bioavailability of therapeutics? Here, we discuss historical and modern drug delivery systems, with the intention of exploring how properties including formulation, administration route and chemical structure influence the ability to achieve extended-release drug release profiles within the body.

Expedient Synthesis of Core Disaccharide Building Blocks from Natural Polysaccharides for Heparan Sulfate Oligosaccharide Assembly.

The complex sulfation motifs of heparan sulfate glycosaminoglycans (HS GAGs) play critical roles in many important biological processes. However, an understanding of their specific functions has been hampered by an inability to synthesize large numbers of diverse, yet defined, HS structures. Herein, we describe a new approach to access the four core disaccharides required for HS/heparin oligosaccharide assembly from natural polysaccharides. The use of disaccharides rather than monosaccharides as minimal precursors greatly accelerates the synthesis of HS GAGs, providing key disaccharide and tetrasaccharide intermediates in about half the number of steps compared to traditional strategies. Rapid access to such versatile intermediates will enable the generation of comprehensive libraries of sulfated oligosaccharides for unlocking the "sulfation code" and understanding the roles of specific GAG structures in physiology and disease.

Probing the Flexibility of the Catalytic Nucleophile in the Lyase Catalytic Pocket of Human DNA Polymerase ß with Unnatural Lysine Analogues.

DNA polymerase ß (Pol ß) is a key enzyme in mammalian base excision repair (BER), contributing stepwise 5'-deoxyribose phosphate (dRP) lyase and "gap-filling" DNA polymerase activities. The lyase reaction is believed to occur via a ß-elimination reaction following the formation of a Schiff base between the dRP group at the pre-incised apurinic/apyrimidinic site and the ε-amino group of Lys72. To probe the steric constraints on the formation and subsequent resolution of the putative Schiff base intermediate within the lyase catalytic pocket, Lys72 was replaced with each of several nonproteinogenic lysine analogues. The modified Pol ß enzymes were produced by coupled in vitro transcription and translation from a modified DNA template containing a TAG codon at the position corresponding to Lys72. In the presence of a misacylated tRNACUA transcript, suppression of the UAG codon in the transcribed mRNA led to elaboration of full length Pol ß having a lysine analogue at position 72. Replacement of the primary nucleophilic amine with a secondary amine in the form of N-methyllysine (4) affected mainly the stability of the Schiff base intermediate and resulted in relatively moderate inhibition of lyase activity and BER. Elongation of the side chain of the catalytic residue by one methylene group, achieved by introduction of homolysine (6) at position 72, apparently shifted the amino group to a position less favorable for Schiff base formation. Interestingly, this effect was attenuated when the side chain was elongated by replacing one side-chain methylene group with a bridging S atom (thialysine, 2). In comparison, replacement of lysine 72 with an analogue having a guanidine moiety in lieu of an ε-amino group (homoarginine, 5) or a sterically constrained secondary amine (piperidinylalanine, 3) led to almost complete suppression of dRP excision activity and the ability of Pol ß to support BER. These results help to define the tolerance of Pol ß to subtle local structural and functional alterations.

Structural features facilitating tumor cell targeting and internalization by bleomycin and its disaccharide.

We have shown previously that the bleomycin (BLM) carbohydrate moiety can recapitulate the tumor cell targeting effects of the entire BLM molecule, that BLM itself is modular in nature consisting of a DNA-cleaving aglycone which is delivered selectively to the interior of tumor cells by its carbohydrate moiety, and that there are disaccharides structurally related to the BLM disaccharide which are more efficient than the natural disaccharide at tumor cell targeting/uptake. Because BLM sugars can deliver molecular cargoes selectively to tumor cells, and thus potentially form the basis for a novel antitumor strategy, it seemed important to consider additional structural features capable of affecting the efficiency of tumor cell recognition and delivery. These included the effects of sugar polyvalency and net charge (at physiological pH) on tumor cell recognition, internalization, and trafficking. Since these parameters have been shown to affect cell surface recognition, internalization, and distribution in other contexts, this study has sought to define the effects of these structural features on tumor cell recognition by bleomycin and its disaccharide. We demonstrate that both can have a significant effect on tumor cell binding/internalization, and present data which suggests that the metal ions normally bound by bleomycin following clinical administration may significantly contribute to the efficiency of tumor cell uptake, in addition to their characterized function in DNA cleavage. A BLM disaccharide-Cy5** conjugate incorporating the positively charged dipeptide d-Lys-d-Lys was found to associate with both the mitochondria and the nuclear envelope of DU145 cells, suggesting possible cellular targets for BLM disaccharide-cytotoxin conjugates.

Cyanotryptophans as Novel Fluorescent Probes for Studying Protein Conformational Changes and DNA-Protein Interaction.

Described herein are the syntheses and photophysical characterization of three novel cyanotryptophans, and their efficient incorporation into proteins as fluorescent probes. Photophysical characteristics indicated that each was significantly brighter and red-shifted in fluorescence emission relative to tryptophan. Each analogue was used to activate a suppressor tRNA transcript and was incorporated with good efficiency into two different positions (Trp22 and Trp74) of Escherichia coli dihydrofolate reductase (ecDHFR). The Trp analogues could be monitored selectively in the presence of multiple native Trp residues in DHFR. 6-CNTrp (A) formed an efficient Förster resonance energy transfer (FRET) pair with l-(7-hydroxycoumarin-4-yl)ethylglycine (HCO, D) at position 17. Further, 6-CNTrp (A) was incorporated into two DNA binding proteins, including the Klenow fragment of DNA polymerase I and an RNA recognition motif (RRM2) of heterogeneous nuclear ribonucleoprotein L-like (hnRNP LL). Using these proteins, we demonstrated the use of FRET involving A as a fluorescence donor and benzo[g]quinazoline-2,4-(1H,3H)-dione 2'-deoxyriboside (Tf) or 4-aminobenzo[g]quinazoline-2-one 2'-deoxyriboside (Cf) as fluorescent acceptors to study the binding interaction of the Klenow fragment with duplex DNA oligomers (labeled with Tf), or the domain-specific association between hnRNP LL and the BCL2 i-motif DNA (labeled with Cf). Thus, the non-natural amino acid could be used as a FRET partner for studying protein-nucleic acid interactions. Together, these findings demonstrate the potential utility of 6-CNTrp (A) as a fluorescence donor for the study of protein conformational events.

The carbamoylmannose moiety of bleomycin mediates selective tumor cell targeting.

Recently, we reported that both bleomycin (BLM) and its disaccharide, conjugated to the cyanine dye Cy5**, bound selectively to cancer cells. Thus, the disaccharide moiety alone recapitulates the tumor cell targeting properties of BLM. Here, we demonstrate that the conjugate of the BLM carbamoylmannose moiety with Cy5** showed tumor cell selective binding and also enhanced cellular uptake in most cancer cell lines. The carbamoyl functionality was required for tumor cell targeting. A dye conjugate prepared from a trivalent cluster of carbamoylmannose exhibited levels of tumor cell binding and internalization significantly greater than those of the simple carbamoylmannose-dye conjugate, consistent with a possible multivalent receptor.

Modified bleomycin disaccharides exhibiting improved tumor cell targeting.

The bleomycins (BLMs) are a family of antitumor antibiotics used clinically for anticancer chemotherapy. Their antitumor selectivity derives at least in part from their ability to target tumor cells, a property that resides in the carbohydrate moiety of the antitumor agent. In earlier studies, we have demonstrated that the tumor cell selectivity resides in the mannose carbamoyl moiety of the BLM saccharide and that both the BLM disaccharide and monosaccharide containing the carbamoyl moiety were capable of the delivery/uptake of a conjugated cyanine dye into cultured cancer cell lines. Presently, the nature of the participation of the carbamoyl moiety has been explored further to provide compounds of utility for defining the nature of the mechanism of tumor cell recognition and uptake by BLM saccharides and in the hope that more efficient compounds could be identified. A library of seven disaccharide-Cy5** dye conjugates was prepared that are structural analogues of the BLM disaccharide. These differed from the natural BLM disaccharide in the position, orientation, and substitution of the carbamoyl group. Studies of these compounds in four matched sets of tumor and normal cell lines revealed a few that were both tumor cell selective and internalized 2-4-fold more efficiently than the natural BLM disaccharide.

The disaccharide moiety of bleomycin facilitates uptake by cancer cells.

The disaccharide moiety is responsible for the tumor cell targeting properties of bleomycin (BLM). While the aglycon (deglycobleomycin) mediates DNA cleavage in much the same fashion as bleomycin, it exhibits diminished cytotoxicity in comparison to BLM. These findings suggested that BLM might be modular in nature, composed of tumor-seeking and tumoricidal domains. To explore this possibility, BLM analogues were prepared in which the disaccharide moiety was attached to deglycobleomycin at novel positions, namely, via the threonine moiety or C-terminal substituent. The analogues were compared with BLM and deglycoBLM for DNA cleavage, cancer cell uptake, and cytotoxic activity. BLM is more potent than deglycoBLM in supercoiled plasmid DNA relaxation, while the analogue having the disaccharide on threonine was less active than deglycoBLM and the analogue containing the C-terminal disaccharide was slightly more potent. While having unexceptional DNA cleavage potencies, both glycosylated analogues were more cytotoxic to cultured DU145 prostate cancer cells than deglycoBLM. Dye-labeled conjugates of the cytotoxic BLM aglycons were used in imaging experiments to determine the extent of cell uptake. The rank order of internalization efficiencies was the same as their order of cytotoxicities toward DU145 cells. These findings establish a role for the BLM disaccharide in tumor targeting/uptake and suggest that the disaccharide moiety may be capable of delivering other cytotoxins to cancer cells. While the mechanism responsible for uptake of the BLM disaccharide selectively by tumor cells has not yet been established, data are presented which suggest that the metabolic shift to glycolysis in cancer cells may provide the vehicle for selective internalization.

Probing of CD4 binding pocket of HIV-1 gp120 glycoprotein using unnatural phenylalanine analogues.

CD4-gp120 interaction is the first step for HIV-1 entry into host cells. A highly conserved pocket in gp120 protein is an attractive target for developing gp120 inhibitors or novel HIV detection tools. Here we incorporate seven phenylalanine derivatives having different sizes and steric conformations into position 43 of domain 1 of CD4 (mD1.2) to explore the architecture of the 'Phe43 cavity' of HIV-1 gp120. The results show that the conserved hydrophobic pocket in gp120 tolerates a hydrophobic side chain of residue 43 of CD protein, which is 12.2 Å in length and 8.0 Å in width. This result provides useful information for developing novel gp120 inhibitors or new HIV detection tools.

Recent Advances in Biomaterials for mRNA Delivery to Immune Cells.

Modulating the function of immune cells by targeting the cells themselves has become a key strategy in immunotherapy for combating various diseases such as cancer, autoimmune disorders, and infectious ailments. The use of mRNA (mRNA) is a powerful tool for transiently inducing protein expression, which is often used for genetic manipulation. However, its inherent instability and inability to precisely reach target cells necessitate the use of biomaterials for safe and effective delivery. Additionally, transfecting immune cells is difficult and complex due to their resistance mechanisms, signaling pathways, and cellular interactions. This review focuses on the recent development of biomaterials for mRNA delivery to immune cells, including lipid nanoparticles and polymeric carriers. It also outlines the challenges of targeting and delivering therapeutic payloads to immune cells, providing commentary and outlook on the design of next-generation materials. Finally, this approach has the potential to significantly enhance the precision and effectiveness of therapeutic interventions for various diseases, shaping the future of mRNA delivery for immune conditions.

Aging-related changes in the mechanical properties of single cells.

Mechanical properties, along with biochemical and molecular properties, play crucial roles in governing cellular function and homeostasis. Cellular mechanics are influenced by various factors, including physiological and pathological states, making them potential biomarkers for diseases and aging. While several methods such as AFM, particle-tracking microrheology, optical tweezers/stretching, magnetic tweezers/twisting cytometry, microfluidics, and micropipette aspiration have been widely utilized to measure the mechanical properties of single cells, our understanding of how aging affects these properties remains limited. To fill this knowledge gap, we provide a brief overview of the commonly used methods to measure single-cell mechanical properties. We then delve into the effects of aging on the mechanical properties of different cell types. Finally, we discuss the importance of studying cellular viscous and viscoelastic properties as well as aging induced by different stressors to gain a deeper understanding of the aging process and aging-related diseases.

Fluorescent biphenyl derivatives of phenylalanine suitable for protein modification.

In a recent study, we demonstrated that structurally compact fluorophores incorporated into the side chains of amino acids could be introduced into dihydrofolate reductase from Escherichia coli (ecDHFR) with minimal disruption of protein structure or function, even when the site of incorporation was within a folded region of the protein. The modified proteins could be employed for FRET measurements, providing sensitive monitors of changes in protein conformation. The very favorable results achieved in that study encouraged us to prepare additional fluorescent amino acids of potential utility for studying protein dynamics. Presently, we describe the synthesis and photophysical characterization of four positional isomers of biphenyl-phenylalanine, all of which were found to exhibit potentially useful fluorescent properties. All four phenylalanine derivatives were used to activate suppressor tRNA transcripts and incorporated into multiple positions of ecDHFR. All phenylalanine derivatives were incorporated with good efficiency into position 16 of ecDHFR and afforded modified proteins that consumed NADPH at rates up to about twice the rate measured for wild type. This phenomenon has been noted on a number of occasions previously and shown to be due to an increase in the off-rate of tetrahydrofolate from the enzyme, altering a step that is normally rate limiting. When introduced into sterically accessible position 49, the four phenylalanine derivatives afforded DHFRs having catalytic function comparable to wild type. The four phenylalanine derivatives were also introduced into position 115 of ecDHFR, which is known to be a folded region of the protein less tolerant of structural alteration. As anticipated, significant differences were noted in the catalytic efficiencies of the derived proteins. The ability of two of the sizable biphenyl-phenylalanine derivatives to be accommodated at position 115 with minimal perturbation of DHFR function is attributed to rotational flexibility about the biphenyl bonds.

Detection of dihydrofolate reductase conformational change by FRET using two fluorescent amino acids.

Two fluorescent amino acids, including the novel fluorescent species 4-biphenyl-l-phenylalanine (1), have been incorporated at positions 17 and 115 of dihydrofolate reductase (DHFR) to enable a study of conformational changes associated with inhibitor binding. Unlike most studies involving fluorescently labeled proteins, the fluorophores were incorporated into the amino acid side chains, and both probes [1 and L-(7-hydroxycoumarin-4-yl)ethylglycine (2)] were smaller than fluorophores typically used for such studies. The DHFR positions were chosen as potentially useful for Förster resonance energy transfer (FRET) measurements on the basis of their estimated separation (17-18 Å) and the expected change in distance along the reaction coordinate. Also of interest was the steric accessibility of the two sites: Glu17 is on the surface of DHFR, while Ile115 is within a folded region of the protein. Modified DHFR I (1 at position 17; 2 at position 115) and DHFR II (2 at position 17; 1 at position 115) were both catalytically competent. However, DHFR II containing the potentially rotatable biphenylphenylalanine moiety at sterically encumbered position 115 was significantly more active than DHFR I. Irradiation of the modified DHFRs at 280 nm effected excitation of 1, energy transfer to 2, and emission by 2 at 450 nm. However, the energy transfer was substantially more efficient in DHFR II. The effect of inhibitor binding was also measured. Trimethoprim mediated concentration-dependent diminution of the emission observed at 450 nm for DHFR II but not for DHFR I. These findings demonstrate that amino acids containing small fluorophores can be introduced into DHFR with minimal disruption of function and in a fashion that enables sensitive monitoring of changes in DHFR conformation.

A retrievable implant for the long-term encapsulation and survival of therapeutic xenogeneic cells.

The long-term function of transplanted therapeutic cells typically requires systemic immune suppression. Here, we show that a retrievable implant comprising a silicone reservoir and a porous polymeric membrane protects human cells encapsulated in it after implant transplantation in the intraperitoneal space of immunocompetent mice. Membranes with pores 1 µm in diameter allowed host macrophages to migrate into the device without the loss of transplanted cells, whereas membranes with pore sizes <0.8 µm prevented their infiltration by immune cells. A synthetic polymer coating prevented fibrosis and was necessary for the long-term function of the device. For >130 days, the device supported human cells engineered to secrete erythropoietin in immunocompetent mice, as well as transgenic human cells carrying an inducible gene circuit for the on-demand secretion of erythropoietin. Pancreatic islets from rats encapsulated in the device and implanted in diabetic mice restored normoglycaemia in the mice for over 75 days. The biocompatible device provides a retrievable solution for the transplantation of engineered cells in the absence of immunosuppression.

Smart Biomaterials: Recent Advances and Future Directions.

Smart biomaterials have the ability to respond to changes in physiological parameters and exogenous stimuli and continue to impact many aspects of modern medicine. Smart materials can promote promising therapies and improve treatment of debilitating diseases. Here, we describe recent advances in the current state-of-the-art design and application of smart biomaterials in tissue engineering, drug delivery systems, medical devices, and immune engineering.