Active control of pharmacokinetics using light-responsive polymer-drug conjugates for boron neutron capture therapy.

In boron neutron capture therapy (BNCT), boron drugs should exhibit high intratumoral boron concentrations during neutron irradiation, while being cleared from the blood and normal organs. However, it is usually challenging to achieve such tumor accumulation and quick clearance simultaneously in a temporally controlled manner. Here, we developed a polymer-drug conjugate that can actively control the clearance of the drugs from the blood. This polymer-drug conjugate is based on a biocompatible polymer that passively accumulates in tumors. Its side chains were conjugated with the low-molecular-weight boron drugs, which are immediately excreted by the kidneys, via photolabile linkers. In a murine subcutaneous tumor model, the polymer-drug conjugate could accumulate in the tumor with the high boron concentration ratio of the tumor to the surrounding normal tissue (∼10) after intravenous injection while a considerable amount remained in the bloodstream as well. Photoirradiation to blood vessels through the skin surface cleaved the linker to release the boron drug in the blood, allowing for its rapid clearance from the bloodstream. Meanwhile, the boron concentration in the tumor which was not photoirradiated could be maintained high, permitting strong BNCT effects. In clinical BNCT, the dose of thermal neutrons to solid tumors is determined by the maximum radiation exposure to normal organs. Thus, our polymer-drug conjugate may enable us to increase the therapeutic radiation dose to tumors in such a practical situation.

Fine tuning of the net charge alternation of polyzwitterion surfaced lipid nanoparticles to enhance cellular uptake and membrane fusion potential.

Lipid nanoparticles (LNPs) coated with functional and biocompatible polymers have been widely used as carriers to deliver oligonucleotide and messenger RNA therapeutics to treat diseases. Poly(ethylene glycol) (PEG) is a representative material used for the surface coating, but the PEG surface-coated LNPs often have reduced cellular uptake efficiency and pharmacological activity. Here, we demonstrate the effect of pH-responsive ethylenediamine-based polycarboxybetaines with different molecular weights as an alternative structural component to PEG for the coating of LNPs. We found that appropriate tuning of the molecular weight around polycarboxybetaine-modified LNP, which incorporated small interfering RNA, could enhance the cellular uptake and membrane fusion potential in cancerous pH condition, thereby facilitating the gene silencing effect. This study demonstrates the importance of the design and molecular length of polymers on the LNP surface to provide effective drug delivery to cancer cells.

The study presents the unique characteristics of small interfering RNA (siRNA)-loaded lipid nanoparticles (LNPs) with different lengths of PGlu(DET-Car), revealing the length of PGlu(DET-Car) critically affects the formation of a stable LNP, the cellular uptake, membrane fusion, and gene silencing abilities.

Self-Folding Macromolecular Drug Carrier for Cancer Imaging and Therapy.

Nano-sized contrast agents (NCAs) hold potential for highly specific tumor contrast enhancement during magnetic resonance imaging. Given the quantity of contrast agents loaded into a single nano-carrier and the anticipated relaxation effects, the current molecular design approaches its limits. In this study, a novel molecular mechanism to augment the relaxation of NCAs is introduced and demonstrated. NCA formation is driven by the intramolecular self-folding of a single polymer chain that possesses systematically arranged hydrophilic and hydrophobic segments in water. Utilizing this self-folding molecular design, the relaxivity value can be elevated with minimal loading of gadolinium complexes, enabling sharp tumor imaging. Furthermore, the study reveals that this NCA can selectively accumulate into tumor tissues, offering effective anti-tumor results through gadolinium neutron capture therapy. The efficacy and versatility of this self-folding molecular design underscore its promise for cancer diagnosis and treatment.

Proposal of recommended experimental protocols for in vitro and in vivo evaluation methods of boron agents for neutron capture therapy.

Recently, boron neutron capture therapy (BNCT) has been attracting attention as a minimally invasive cancer treatment. In 2020, the accelerator-based BNCT with L-BPA (Borofalan) as its D-sorbitol complex (Steboronine®) for head and neck cancers was approved by Pharmaceutical and Medical Devices Agency for the first time in the world. As accelerator-based neutron generation techniques are being developed in various countries, the development of novel tumor-selective boron agents is becoming increasingly important and desired. The Japanese Society of Neutron Capture Therapy believes it is necessary to propose standard evaluation protocols at each stage in the development of boron agents for BNCT. This review summarizes recommended experimental protocols for in vitro and in vivo evaluation methods of boron agents for BNCT based on our experience with L-BPA approval.

pH-responsive polyzwitterion covered nanocarriers for DNA delivery.

The success of gene therapy relies on gene nanocarriers to achieve therapeutic effects in vivo. Surface shielding of poly(ethylene glycol) (PEG), known as PEGylation, onto gene delivery carriers is a predominant strategy for extending blood circulation and improving therapeutic outcomes in vivo. Nevertheless, PEGylation frequently compromises the transfection efficiency by decreasing the interactions with the cellular membrane of the targeted cells, thereby preventing the cellular uptake and the subsequent endosomal escape. Herein, we developed a stepwise pH-responsive polyplex micelle for the plasmid DNA delivery with the surface covered by ethylenediamine-based polycarboxybetaines. This polyplex micelle switched its surface charge from neutral at pH 7.4 to positive at tumorous and endo-/lysosomal pH (i.e., pH 6.5 and 5.5, respectively), thus enhancing the cellular uptake and facilitating the endosomal escape toward efficient gene transfection. Additionally, the polyplex micelle demonstrated prolonged blood circulation as well as enhanced tumor accumulation, leading to highly effective tumor growth suppression by delivering an antiangiogenic gene. These results suggest the usefulness of a pH-responsive charge-switchable shell polymer on the surface of the polyplex micelle for the efficient nucleic acid delivery.

Polymeric ligands comprising sulfur-containing amino acids for targeting tumor-associated amino acid transporters.

Various cancer cells overexpress L-type amino acid transporter 1 (LAT1) to take up a large number of neutral amino acids such as phenylalanine and methionine, and LAT1 transporter should be a promising target for cancer diagnosis and therapy. However, only a few studies reported drug delivery systems targeting LAT1 probably due to limited knowledge about the interaction between LAT1 and its substrate. Here, we developed polymers having methionine (Met)- or cysteine (Cys)-like structures on their side chains to examine their affinity with LAT1. While both the Met- and Cys-modified polymers exhibited efficient cellular uptake selectively in cancer cells, the Met-modified polymers exhibited higher cellular uptake efficiency in an LAT1-selective manner than the Cys-modified polymers. In the in vivo study, the intraperitoneally injected Met-modified polymers showed appreciable tumor-selective accumulation in the peritoneal dissemination model, and importantly, Met-modified polymers conjugated with photosensitizers exhibited significant therapeutic effects upon photoirradiation with reduced photochemical damage to normal organs. Our results may provide important knowledge about the polymer-LAT1 interaction, and the Met-modified polymers should offer a new concept for designing LAT1-targeting drug delivery systems.

Thermo-Responsive Polymer-siRNA Conjugates Enabling Artificial Control of Gene Silencing around Body Temperature.

PURPOSE: Controlling small interfering RNA (siRNA) activity by external stimuli is useful to exert a selective therapeutic effect at the target site. This study aims to develop a technology to control siRNA activity in a thermo-responsive manner, which can be utilized even at temperatures close to body temperature. METHODS: siRNA was conjugated with a thermo-responsive copolymer that was synthesized by copolymerization of N-isopropylacrylamide (NIPAAm) and hydrophilic N,N-dimethylacrylamide (DMAA) to permit thermally controlled interaction between siRNA and an intracellular gene silencing-related protein by utilizing the coil-to-globule phase transition of the copolymer. The composition of the copolymer was fine-tuned to obtain lower critical solution temperature (LCST) around body temperature, and the phase transition behavior was evaluated. The cellular uptake and gene silencing efficiency of the copolymer-siRNA conjugates were then investigated in cultured cells. RESULTS: The siRNA conjugated with the copolymer with LCST of 38.0°C exhibited ~ 11.5 nm of the hydrodynamic diameter at 37°C and ~ 9.8 nm of the diameter at 41°C, indicating the coil-globule transition above the LCST. In line with this LCST behavior, its cellular uptake and gene silencing efficiency were enhanced when the temperature was increased from 37°C to 41°C. CONCLUSION: By fine-tuning the LCST behavior of the copolymer that was conjugated with siRNA, siRNA activity could be controlled in a thermo-responsive manner around the body temperature. This technique may offer a promising approach to induce therapeutic effects of siRNA selectively in the target site even in the in vivo conditions.

Polymeric iron chelators for enhancing 5-aminolevulinic acid-induced photodynamic therapy.

5-Aminolevulinic acid (5-ALA) is an amino acid that can be metabolized into a photosensitizer, protoporphyrin IX (PpIX) selectively in a tumor cell, permitting minimally invasive photodynamic diagnosis/therapy. However, some malignant tumor cells have excess intracellular labile iron and facilitate the conversion of PpIX into heme, which compromises the therapeutic potency of 5-ALA. Here, we examined the potential of chelation of such unfavorable intratumoral labile iron in photodynamic therapy (PDT) with 5-ALA hydrochloride, using polymeric iron chelators that we recently developed. The polymeric iron chelator efficiently inactivated the intracellular labile iron in cultured cancer cells and importantly enhanced the accumulation of PpIX, thereby improving the cytotoxicity upon photoirradiation. Even in in vivo study with subcutaneous tumor models, the polymeric iron chelator augmented the intratumoral accumulation of PpIX and the PDT effect. This study suggests that our polymeric iron chelator could be a tool for boosting the effect of 5-ALA-induced PDT by modulating tumor microenvironment.

Cancerous pH-responsive polycarboxybetaine-coated lipid nanoparticle for smart delivery of siRNA against subcutaneous tumor model in mice.

Lipid nanoparticles (LNPs) have been commonly used as a vehicle for nucleic acids, such as small interfering RNA (siRNA); the surface modification of LNPs is one of the determinants of their delivery efficiency especially in systemic administration. However, the applications of siRNA-encapsulated LNPs are limited due to a lack effective systems to deliver to solid tumors. Here, we report a smart surface modification using a charge-switchable ethylenediamine-based polycarboxybetaine for enhancing tumor accumulation via interaction with anionic tumorous tissue constituents due to selective switching to cationic charge in response to cancerous acidic pH. Our polycarboxybetaine-modified LNP could enhance cellular uptake in cancerous pH, resulting in facilitated endosomal escape and gene knockdown efficiency. After systemic administration, the polycarboxybetaine-modified LNP accomplished high tumor accumulation in SKOV3-luc and CT 26 subcutaneous tumor models. The siPLK-1-encapsulated LNP thereby accomplished significant tumor growth inhibition. This study demonstrates a promising potential of the pH-responsive polycarboxybetaine as a material for modifying the surface of LNPs for efficient nucleic acid delivery.

Changeable net charge on nanoparticles facilitates intratumor accumulation and penetration.

The Enhanced Permeability and Retention (EPR) effect is a golden strategy for the nanoparticle (NP)-based targeting of solid tumors, and the surface property of NPs might be a determinant on their targeting efficiency. Poly(ethylene glycol) (PEG) is commonly used as a shell material; however, it has been pointed out that PEG-coated NPs may exhibit accumulation near tumor vasculature rather than having homogenous intratumor distribution. The PEG shell plays a pivotal role on prolonged blood circulation of NPs but potentially impairs the intratumor retention of NPs. In this study, we report on a shell material to enhance tumor-targeted delivery of NPs by maximizing the EPR effect: polyzwitterion based on ethylenediamine-based carboxybetaine [PGlu(DET-Car)], which shows the changeable net charge responding to surrounding pH. The net charge of PGlu(DET-Car), is neutral at physiological pH 7.4, allowing it to exhibit a stealth property during the blood circulation; however, it becomes cationic for tissue-interactive performance under tumorous acidic conditions owing to the stepwise protonation behavior of ethylenediamine. Indeed, the PGlu(DET-Car)-coated NPs (i.e., gold NPs in the present study) exhibited prolonged blood circulation and remarkably enhanced tumor accumulation and retention than PEG-coated NPs, achieving 32.1% of injected dose/g of tissue, which was 4.2 times larger relative to PEG-coated NPs. Interestingly, a considerable portion of PGlu(DET-Car)-coated NPs clearly penetrated into deeper tumor sites and realized the effective accumulation in hypoxic regions, probably because the cationic net charge of PGlu(DET-Car) is augmented in more acidic hypoxic regions. This study suggests that the changeable net charge on the NP surface in response to tumorous acidic conditions is a promising strategy for tumor-targeted delivery based on the EPR effect.

Quantitative Analysis of Acoustic Pressure for Sonophoresis and Its Effect on Transdermal Penetration.

Ultrasound facilitates the penetration of macromolecular compounds through the skin and offers a promising non-invasive technique for transdermal delivery. However, technical difficulties in quantifying ultrasound-related parameters have restricted further analysis of the sonophoresis mechanism. In this study, we devise a bolt-clamped Langevin transducer-based sonophoresis device that enables us to measure with a thin lead zirconate titanate (PZT) sensor. One-dimensional acoustic theory accounting for wave interaction at the skin interface indicates that the acoustic pressure and cavitation onset on the skin during sonophoresis are sensitive to the subcutaneous support, meaning that there is a strong need to perform the pressure measurement in an experimental environment replacing the human body. From a series of the experiments with our new device, the transdermal penetration of polystyrene, silica and gold nanoparticles is found to depend on the size and material of the particles, as well as the hardness of the subcutaneous support material. We speculate from the acoustic pressure measurement that the particles' penetration results from the mechanical action of cavitation.

Sequentially Self-Assembled Nanoreactor Comprising Tannic Acid and Phenylboronic Acid-Conjugated Polymers Inducing Tumor-Selective Enzymatic Activity.

The construction of enzyme delivery systems, which can control enzymatic activity at a target site, is important for efficient enzyme-prodrug therapy/diagnosis. Herein we report a facile technique to construct a systemically applicable ß-galactosidase (ß-Gal)-loaded ternary complex comprising tannic acid (TA) and phenylboronic acid-conjugated polymers through sequential self-assembly in aqueous solution. At physiological conditions, the ternary complex exhibited a hydrodynamic diameter of ∼40 nm and protected the loaded ß-Gal from unfavorable degradation by proteinase. Upon cellular internalization, the ternary complex recovered ß-Gal activity by releasing the loaded ß-Gal. The intravenously injected ternary complex thereby delivered ß-Gal to the target tumor in a subcutaneous tumor model and exerted enhanced and selective enzymatic activity at the tumor site. Sequential self-assembly with TA and phenylboronic acid-conjugated polymers may offer a novel approach for enzyme-prodrug theragnosis.

Polymeric Iron Chelators Enhancing Pro-Oxidant Antitumor Efficacy of Vitamin C by Inhibiting the Extracellular Fenton Reaction.

Intravenously injected high-dose vitamin C (VC) induces extracellular H2O2, which can penetrate into the tumor cells and suppress tumor growth. However, extracellular labile iron ions in the tumor decompose H2O2 via the Fenton reaction, limiting the therapeutic effect. In this regard, we recently developed a polymeric iron chelator that can inactivate the intratumoral labile iron ions. Here, we examined the effect of our polymeric iron chelator on the high-dose VC therapy in in vitro and in vivo. In the in vitro study, the polymeric iron chelator could inactivate the extracellular labile iron ions and prevent the unfavorable decomposition of VC-induced H2O2, augmenting pro-oxidative damage to DNA and inducing apoptosis in cultured cancer cells. Even in the in vivo study, the polymeric iron chelator significantly improved the antitumor effect of VC in subcutaneous DLD-1 and CT26 tumors in mice, while conventional iron chelators could not. This work indicates the importance of modulating tumor-associated iron ions in the high-dose VC therapy and should contribute to a better understanding of its mechanism.

Differential Effect of Azetidine Substitution in Firefly Luciferin Analogues.

Replacing an N,N-dimethylamino group in a classical fluorophore with a four membered azetidine ring provides an improved luminescence quantum yield. Herein, we extended this strategy to bioluminescent firefly luciferin analogues and evaluated its general validity. For this purpose, four types of luciferin cores were employed, and a total of eight analogues were evaluated. Among these analogues, unexpectedly, only the benzothiazole core analogue benefited from an azetidine substitution and showed enhanced bioluminescence. In addition, fluorescence measurements revealed that an azetidine substitution improved the fluorescence quantum yield by 2.3-times compared to a N,N-dimethylamino group. These findings clarify the differential effects of azetidine substituents in luciferins and present one possible strategy for enhancing photon output in benzothiazole type luciferins through a synthetic approach.

Synthesis of near-infrared absorbing triangular Au nanoplates using biomineralisation peptides.

Triangular Au nanoplates (TrAuNPls) possessing strong plasmonic properties can be used as photothermal agents in cancer therapy. However, the controlled preparation of such morphologies typically requires harsh synthetic conditions. Biomolecules offer an alternative route to developing biocompatible synthetic protocols. In particular, peptides offer a novel route for inorganic synthesis under ambient conditions. Herein, using the previously isolated peptide, ASHQWAWKWE, for Au nanoparticle (AuNP) synthesis, the conditions for preparing TrAuNPls via a one-pot synthetic process of mixing HAuCl4 and peptides at room temperature were investigated to effectively obtain particles possessing near-infrared absorbance for non-invasive optical diagnosis and phototherapy. By adjusting the peptide concentration, the size and property of TrAuNPls were controlled under neutral pH conditions. The synthesised particles showed potential as photothermal therapeutic agents in vitro. In addition, peptide characterisation using B3 derivatives revealed the importance of the third amino acid histidine in morphological regulation and potential circular Au nanoplates (AuNPl) synthesis with ASEQWAWKWE and ASAQWAWKWE peptides. These findings provide not only an easy and green synthetic method for TrAuNPls and circular AuNPls, but also some insight to help elucidate the regulation of peptide-based nanoparticle synthesis for use in cancer therapy. STATEMENT OF SIGNIFICANCE: Biological molecules have received increasing attention as a vehicle to synthesise inorganic materials with specific properties under ambient conditions; particularly, short peptides have the potential to control the synthesis of nanoscale materials with tailored functions. Here, the application of a previously isolated peptide was assessed in synthesising Au nanoparticles containing decahedral and triangular nanoplates with near-infrared absorbance. The size and absorbance peaks of the triangular nanoplates observed were peptide concentration-dependent. In addition, these fine-tuned triangular nanoplates exhibited potential as a phototherapeutic agent. Moreover, the peptide derivatives indicated the possibility of synthesising circular nanoplates. These findings may offer insight into development of new techniques for synthesising functional nanoparticles having biological applications using non-toxic molecules under mild conditions stituted in the original B3 peptide is underlined.

Fructose-functionalized polymers to enhance therapeutic potential of p-boronophenylalanine for neutron capture therapy.

In boron neutron capture therapy (BNCT), boron drugs should accumulate selectively within a tumor and be quickly cleared from blood and normal organs. However, it is usually challenging to achieve the efficient tumor accumulation and the quick clearance simultaneously. Here we report the complex composed of a fructose-modified poly(ethylene glycol)-poly(l-lysine) block copolymer and p-boronophenylalanine, termed PEG-P[Lys/Lys(fructose)]-BPA, as a boron delivery system permitting selective accumulation within the target tumor with quick clearance from normal organs as well as blood. Our PEG-P[Lys/Lys(fructose)]-BPA could be internalized into tumor cells through LAT1 amino acid transporter-mediated endocytosis and retain in the targeted cells, thereby accomplishing more efficient accumulation and retention in a subcutaneous tumor than clinically used fructose-BPA complexes. Importantly, the moderately cationic property of the polymer facilitated renal clearance and PEG-P[Lys/Lys(fructose)]-BPA exhibited high accumulation contrast between the target tumor and the blood/normal organ. Finally, upon thermal neutron irradiation, PEG-P[Lys/Lys(fructose)]-BPA significantly inhibited the tumor growth in mice. PEG-P[Lys/Lys(fructose)]-BPA may be a promising boron delivery system for BNCT.

Potential urinary monitoring of the enhanced permeability and retention effect using MMP-2-responsive poly(ethylene glycol) derivatives.

The enhanced permeability and retention (EPR) effect is fundamental to tumor-targeted drug delivery using nanoparticles. However, recent studies reported heterogeneity of the EPR effect, and companion diagnostics are considered to be key to predicting and optimizing the benefits of the EPR effect. Here, as a new material to simply endow the function of companion diagnostics to nanoparticles, we designed a poly(ethylene glycol) (PEG) derivative conjugated with low molecular fluorescent dye through synthetic substrate linker that can be cleaved in response to MMP-2, which is overexpressed in tumor extracellular matrix. Upon tumor accumulation, the low molecular fluorescent dye is released from the PEG and quickly excreted to urine, thereby reporting its tumor accumulation level as a fluorescent signal in the urine. In this study, this urinary reporter was conjugated with albumin, and the functionalized albumin exhibited efficient accumulation in various tumors. Importantly, the functionalized albumin exhibited significantly higher excretion of the fluorescent dye in the urine in mice with tumors compared with those without tumors. The PEG derivatives proposed in this study may be a promising tool to predict the EPR effect in individual cancer patients.

Systemically Applicable Glutamine-Functionalized Polymer Exerting Multivalent Interaction with Tumors Overexpressing ASCT2.

Transporter ASCT2, which predominantly imports glutamine (Gln), is overexpressed in a variety of cancer cells, and targeting ASCT2 is expected to be a promising approach for tumor diagnosis and therapy. In this work, we designed a series of glutamine-modified poly(l-lysine) (PLys(Gln)) homopolymers and PEG-PLys(Gln) block copolymers and investigated their tumor-targeting abilities. With increasing degree of polymerization in the PLys(Gln) homopolymers, their cellular uptake was gradually enhanced through multivalent interactions with ASCT2. The performance of PEG-PLys(Gln) in blood circulation and tumor accumulation could be controlled by tuning of the molecular weight of PEG. Our results highlight the utility of molecular recognition in ASCT2/PLys(Gln) for tumor targeting through systemic administration.

Iron chelation cancer therapy using hydrophilic block copolymers conjugated with deferoxamine.

Cancer cells have high iron requirements due to their rapid growth and proliferation. Iron depletion using iron chelators has a potential in cancer treatment. Previous studies have demonstrated that deferoxamine (DFO) specifically chelates Fe(III) and exhibited antitumor activity in clinical studies. However, its poor pharmacokinetics has limited the therapeutic potential and practical application. Although polymeric iron chelators have been developed to increase the blood retention, none of previous studies has demonstrated their potential in iron chelation cancer therapy. Here, we developed polymeric DFO by the covalent conjugation of DFO to poly(ethylene glycol)-poly(aspartic acid) (PEG-PAsp) block copolymers. The polymeric DFO exhibited iron-chelating ability comparable with free DFO, thereby arresting cell cycle and inducing apoptosis and antiproliferative activity. After intravenous administration, the polymeric DFO showed marked increase in blood retention and tumor accumulation in subcutaneous tumor models. Consequently, polymeric DFO showed significant suppression of the tumor growth compared with free DFO. This study reveals the first success of the design of polymeric DFO for enhancing iron chelation cancer therapy.

Photodynamic therapy using LCST polymers exerting pH-responsive isothermal phase transition.

In photodynamic therapy (PDT), the inherent physicochemical properties of a photosensitizer (PS) critically affect its biodistribution and therapeutic outcome as well as side effect. Here, we developed a PS-polymer conjugate displaying isothermal hydrophilic-to-hydrophobic phase transition in response to tumorous acidic pH. The polymer backbone was poly(N-isopropylacrylamide (NIPAAm)/2-aminoisoprpylacrylamide (AIPAAm)) (P(NIPAAm/AIPAAm)), which shows lower critical solution temperature (LCST) of 30 °C. The amine groups in its side chains were converted to hydrophilic acid-labile 2-propionic-3-methylmaleic (PMM) amides, forming poly(NIPAAm/AIPAAm-PMM). The conjugation of PMM moieties drastically increased the LCST of the polymer to 40 °C and displayed hydrophilic character to minimalize unspecific interaction of PS-P(NIPAAm/AIPAAm-PMM) in bloodstream, diminishing potential photosensitivity. The detachment of PMM at tumorous pH lowered the LCST to that of original P(NIPAAm/AIPAAm), permitting hydrophilic-to-hydrophobic transition at a physiological temperature (37 °C). This pH-responsive isothermal phase transition facilitated interaction with the cultured cancer cells, accomplishing 8.1 times-enhanced cellular uptake and strong phototoxicity in a tumorous pH-selective manner. Even in subcutaneous tumor models, our polymer conjugates exhibited efficient tumor accumulation and significantly augmented PDT effect without inducing unfavorable photochemical toxicity to the skin. This study offers a novel concept of PS delivery systems targeting tumorous pH by the use of isothermal phase transition.