Glucose transporter 1-mediated vascular translocation of nanomedicines enhances accumulation and efficacy in solid tumors.

Nanomedicine modification with ligands directed to receptors on tumor blood vessels has the potential for selectively enhancing nanomedicine accumulation in malignant tissues by overcoming the vascular barrier of tumors. Nevertheless, the development of broadly applicable ligand approaches capable of promoting the transvascular transport of nanomedicines in a wide spectrum of tumors has been elusive so far. By considering the indispensable and persistent glycolytic fueling of tumors, we developed glucose-installed polymeric micelles loading cisplatin (Gluc-CDDP/m) targeting the glucose transporter 1 (GLUT1), which is overexpressed in most tumors and present on vascular endothelial cells, toward improving the delivery efficiency and therapeutic efficacy. The design of the glucose ligands on Gluc-CDDP/m was engineered to control the conjugation via the carbon 6 of the glucose moieties, as well as the ligand density on the poly (ethylene glycol) (PEG) shell of the micelles. The series of micelles was then studied in vitro and in vivo against GLUT1-high human squamous cell carcinoma of the head and neck OSC-19 cells and GLUT1-low human glioblastoma-astrocytoma U87MG cells. Our results showed that precisely tuning the micelles to have glucose ligands on 25% of their PEG chains increased the efficacy against the tumors by significantly enhancing the tumor accumulation, even in GLUT1-low U87MG tumors. The enhancement of the intratumoral levels of these micelles was hindered by concomitant administration of glucose, or the GLUT1 inhibitor STF-31, confirming a GLUT1/glucose-mediated increment of the accumulation. Intravital confocal laser scanning microscopy imaging of tumor tissues further demonstrated the rapid extravasation and penetration of Gluc-CDDP/m in OSC-19 tumors compared to non-targeted CDDP/m. These findings indicate GLUT1-targeting as a promising approach for overcoming the vascular barrier and boosting the delivery of nanomedicine in tumors.

Enhanced Intracellular Delivery of siRNA by Controlling ATP-Responsivity of Phenylboronic Acid-Functionalized Polyion Complex Micelles.

Intracellular delivery of small interfering RNA (siRNA) is a long-standing challenge in oligonucleotide therapeutics. Herein, adenosine triphosphate (ATP)-responsive polyion complex micelles assembled from poly(ethylene glycol)-block-poly(l-lysine) (PEG-PLys) bearing 4-carboxy-3-fluorophenylboronic acid (FPBA) moiety in the PLys side chains (FPBA micelle) for the delivery of cholesterol-modified siRNA (Chol-siRNA) are described. The pKa of FPBA moiety is 7.2 and, therefore, it exists in equilibrium between negatively charged tetravalent and noncharged hydrophobic trivalent forms in physiological pH conditions. Each form cooperatively stabilizes the micelle in distinct modes, that is, a covalent ester-linkage between charged boronate and ribose functionality at 3' ends of Chol-siRNA and a hydrophobic interaction between noncharged boronic acid and Chol-siRNA. When exposed to ATP at a concentration associated with the intracellular environment, the Chol-siRNA/boronate linkage is readily cleaved to facilitate the release of Chol-siRNA into cytoplasm. In order to further optimize this switching capability, the effect of FPBA modification rate is studied for the resulting ATP-responsive behavior of the micelles. As a result, the range of 23-35% in the modification rate is found suitable to maximize the gene silencing efficiency, demonstrating the potential of the FPBA-modified micelles as ATP-responsive smart siRNA carrier systems.

Polyplex Micelles with Phenylboronate/Gluconamide Cross-Linking in the Core Exerting Promoted Gene Transfection through Spatiotemporal Responsivity to Intracellular pH and ATP Concentration.

Polyplexes as gene delivery carriers require integrated functionalities to modulate intracellular trafficking for efficient gene transfection. Herein, we developed plasmid DNA (pDNA)-loaded polyplex micelles (PMs) from poly(ethylene glycol)-based block catiomers derivatized with 4-carboxy-3-fluorophenylboronic acid (FPBA) and d-gluconamide to form pH- and ATP-responsive cross-linking in the core. These PMs exhibited robustness in the extracellular milieu and smooth endosomal escape after cellular uptake, and they facilitated pDNA decondensation triggered by increased ATP concentration inside of the cell. Laser confocal microscopic observation revealed that FPBA installation enhanced the endosomal escapability of the PMs; presumably, this effect resulted from the facilitated endo-/lysosomal membrane disruption triggered by the released block catiomers with hydrophobic FPBA moieties in the side chain from the PM at lower pH condition of endo-/lysosomes. Furthermore, the profile of intracellular pDNA decondensation from the PMs was monitored using Förster resonance energy transfer measurement by flow cytometry; these observations confirmed that PMs optimized for ATP-responsivity exerted effective intracellular decondensation of loaded pDNA to attain promoted gene transfection.

Multilayered polyion complexes with dissolvable silica layer covered by controlling densities of cRGD-conjugated PEG chains for cancer-targeted siRNA delivery.

Surface functionalization of nanoparticles is a crucial factor for nanoparticle-mediated drug and nucleic acid delivery. Particularly, the density of targeting ligands on nanoparticle significantly affects the affinity of nanoparticles to specific cellular surface (or receptor) through the multivalent binding effect. Herein, multilayered polyion complexes (mPICs) are prepared to possess varying densities of cyclic RGD peptide (cRGD) ligands for cancer-targeted small interfering RNA (siRNA) delivery. A template PIC is first prepared by mixing siRNAs with homo catiomers of N-substituted polyaspartamide bearing tetraethylenepentamine (PAsp(TEP)) in aqueous solution, followed by silica-coating through silicate condensation reaction. Then, silica-coated PICs (sPICs) are further covered with block catiomers of PAsp(TEP) and poly(ethylene glycol) (PEG) equipped with cRGD ligand. Successful preparation of targeted mPICs is confirmed from the changes in size and ζ-potential and the elemental analysis by transmission electron microscopy. Notably, the number of cRGD ligands per mPIC is regulated by altering the silicate concentration upon preparation of sPICs, which is confirmed by fluorescence correlation spectroscopy using fluorescent-labeled block catiomers. Ultimately, the targeted mPICs with a higher number of cRGD ligands demonstrate more efficient cellular uptake in cultured cancer cells, leading to enhanced gene silencing activity.

Synthetic Polyamines to Regulate mRNA Translation through the Preservative Binding of Eukaryotic Initiation Factor 4E to the Cap Structure.

Polyion complexes (PICs) of mRNA with synthetic polyamines are receiving increasing attention as mRNA delivery vehicles, and the search for polyamine structure maximizing the translational efficiency of complexed mRNA becomes a critical research topic. Herein, we discovered that fine-tuning of the protonation status of synthetic polyamines can regulate mRNA translation through the preservative binding of eukaryotic initiation factor 4E to m(7)GpppN (cap structure) on the 5' end of mRNA. A series of polyamines with varied numbers of aminoethylene repeats in their side chains were prepared by an aminolysis reaction of poly(ß-benzyl-l-aspartate) and paired with mRNA to form PICs. PICs formed from polyamines with higher numbers of aminoethylene repeats preserved the original translational efficiency to naked mRNA, whereas the efficiency significantly dropped by decreasing the number of aminoethylene repeats in the polyamines. Immunoprecipitation assays using anti-eIF4E antibodies revealed that the binding affinity of eIF4E to the cap structure of mRNA in the PIC was sensitive to the number of charged aminoethylene repeats in the polyamine side chain and was strongly correlated with their translational efficiency. These results indicate that the fine-tuning of the polyamine structure plays a critical role in maximizing the translational efficiency of mRNA in the PICs having potential utility as mRNA delivery vehicles.

Systemic delivery of messenger RNA for the treatment of pancreatic cancer using polyplex nanomicelles with a cholesterol moiety.

Systemic delivery of messenger RNA (mRNA) is technically challenging because mRNA is highly susceptible to enzymatic degradation in the blood circulation. In this study, we used a nanomicelle-based platform, prepared from mRNA and poly(ethylene glycol) (PEG)-polycation block copolymers. A cholesterol (Chol) moiety was attached to the ω-terminus of the block copolymer to increase the stability of the nanomicelle by hydrophobic interaction. After in vitro screening, polyaspartamide with four aminoethylene repeats in its side chain (PAsp(TEP)) was selected as the cationic segment of the block copolymer, because it contributes to enhance nuclease resistance and high protein expression from the mRNA. After intravenous injection, PEG-PAsp(TEP)-Chol nanomicelles showed significantly enhanced blood retention of mRNA in comparison to nanomicelles without Chol. We used the nanomicelles for treating intractable pancreatic cancer in a subcutaneous inoculation mouse model through the delivery of mRNA encoding an anti-angiogenic protein (sFlt-1). PEG-PAsp(TEP)-Chol nanomicelles generated efficient protein expression from the delivered mRNA in tumor tissue, resulting in remarkable inhibition of the tumor growth, whereas nanomicelles without Chol failed to show a detectable therapeutic effect. In conclusion, the stabilized nanomicelle system led to the successful systemic delivery of mRNA in therapeutic application, holding great promise for the treatment of various diseases.

Enhanced target recognition of nanoparticles by cocktail PEGylation with chains of varying lengths.

Monodispersed gold nanoparticles (AuNPs) were simultaneously decorated with lactosylated and non-modified shorter poly(ethylene glycol)s (PEGs) to enhance their target recognition. The decoration with sufficiently shorter PEGs dramatically enhanced the multivalent binding ability of lactosylated AuNPs to the lectin-fixed surface, possibly due to the enhanced mobility of the ligands via the spacer effect generated by the shorter PEG chains.

Intracellular Delivery of Charge-Converted Monoclonal Antibodies by Combinatorial Design of Block/Homo Polyion Complex Micelles.

Direct intracellular delivery of antibodies has gained much attention, although only a few agents have been developed, and none of them has reached clinical stages. The main obstacles here are the insufficient characteristics of delivery systems including stability and appropriate ability for intracellular antibody release. We tailored the structure of polyion complex (PIC) micelles by loading transiently charge-converted antibody derivatives for achieving enhanced stability, delivery to cytosol, and precise antigen recognition inside cells. Citraconic anhydride was used for the charge conversion of the antibody; the optimized degree of modification was identified to balance the stability of PIC micelles in the extracellular compartment and prompt pH-triggered disintegration after their translocation into the acidic endosomal compartment of target cells. The use of a mixture of homo- and block-catiomers in an appropriate ratio to construct PIC micelles substantially enhanced the endosomal escaping efficacy of the loaded antibody, leading to improved recognition of intracellular antigens.

Polyplex Micelles with Double-Protective Compartments of Hydrophilic Shell and Thermoswitchable Palisade of Poly(oxazoline)-Based Block Copolymers for Promoted Gene Transfection.

Improving the stability of polyplex micelles under physiological conditions is a critical issue for promoting gene transfection efficiencies. To this end, hydrophobic palisade was installed between the inner core of packaged plasmid DNA (pDNA) and the hydrophilic shell of polyplex micelles using a triblock copolymer consisting of hydrophilic poly(2-ethyl-2-oxazoline), thermoswitchable amphiphilic poly(2-n-propyl-2-oxazoline) (PnPrOx) and cationic poly(L-lysine). The two-step preparation procedure, mixing the triblock copolymer with pDNA below the lower critical solution temperature (LCST) of PnPrOx, followed by incubation above the LCST to form a hydrophobic palisade of the collapsed PnPrOx segment, induced the formation of spatially aligned hydrophilic-hydrophobic double-protected polyplex micelles. The prepared polyplex micelles exhibited significant tolerance against attacks from nuclease and polyanions compared to those without hydrophobic palisades, thereby promoting gene transfection. These results corroborated the utility of amphiphilic poly(oxazoline) as a molecular thermal switch to improve the stability of polyplex gene carriers relevant for physiological applications.

Messenger RNA-based therapeutics for the treatment of apoptosis-associated diseases.

Gene therapy is a promising approach for treating diseases that are closely associated with excessive apoptosis, because the gene can effectively and sustainably introduce anti-apoptotic factors into cells. However, DNA delivery poses the risk of random genomic integration, leading to overexpression of the delivered gene and cancer development. Messenger RNA (mRNA) can evade integration events in target cells. We examined the use of mRNA-based therapeutics for introducing anti-apoptotic factors by using a mouse model of fulminant hepatitis. For introducing mRNA into the liver, a synthesised polymer-based carrier of polyplex nanomicelles was used for hydrodynamic intravenous injection. Using GFP as a reporter, we demonstrate that mRNA delivery induced efficient protein expression in almost 100% of liver cells, while plasmid DNA (pDNA) delivery provided a smaller percentage of GFP-positive cells. Analyses using Cy5-labelled mRNA and pDNA revealed that efficient expression by mRNA was attributed to a simple intracellular mechanism, without the need for nuclear entry. Consistent with this observation, Bcl-2 mRNA was more effective on reducing apoptosis in the liver of mice with fulminant hepatitis than Bcl-2 pDNA. Therefore, mRNA-based therapeutics combined with an effective delivery system such as polyplex nanomicelles is a promising treatment for intractable diseases associated with excessive apoptosis.

Development of Biodegradable Polycation-Based Inhalable Dry Gene Powders by Spray Freeze Drying.

In this study, two types of biodegradable polycation (PAsp(DET) homopolymer and PEG-PAsp(DET) copolymer) were applied as vectors for inhalable dry gene powders prepared by spray freeze drying (SFD). The prepared dry gene powders had spherical and porous structures with a 5~10-µm diameter, and the integrity of plasmid DNA could be maintained during powder production. Furthermore, it was clarified that PEG-PAsp(DET)-based dry gene powder could more sufficiently maintain both the physicochemical properties and in vitro gene transfection efficiencies of polyplexes reconstituted after powder production than PAsp(DET)-based dry gene powder. From an in vitro inhalation study using an Andersen cascade impactor, it was demonstrated that the addition of l-leucine could markedly improve the inhalation performance of dry powders prepared by SFD. Following pulmonary delivery to mice, both PAsp(DET)- and PEG-PAsp(DET)-based dry gene powders could achieve higher gene transfection efficiencies in the lungs compared with a chitosan-based dry gene powder previously reported by us.

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 µm diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

Modulated protonation of side chain aminoethylene repeats in N-substituted polyaspartamides promotes mRNA transfection.

Fine-tuning of chemical structures of polycation-based carriers (polyplexes) is an attractive strategy for safe and efficient mRNA transfaction. Here, mRNA polyplexes comprising N-substituted polyaspartamides with varied numbers of side chain aminoethylene repeats were constructed, and their transfection ability against human hepatoma cells was examined. Transfection efficacy clearly correlated with the number of aminoethylene repeats: polyplexes with odd number repeats (PA-Os) produced sustained increases in mRNA expression compared with those with even number repeats (PA-Es). This predominant efficacy of PA-Os over PA-Es was contradictory to our previous findings for pDNA polyplexes prepared from the same N-substituted polyaspartamides, that is, PA-Es revealed superior transfection efficacy of pDNA than PA-Os. Intracellular FRET analysis using flow cytometry and polyplex tracking under confocal laser scanning microscopy revealed that overall transfection efficacy was determined through the balance between endosomal escaping capability and stability of translocated mRNA in cytoplasm. PA-Es efficiently transported mRNA into the cytoplasm. However, their poor cytoplasmic stability led to facile degradation of mRNA, resulting in a less durable pattern of transfection. Alternatively, PA-Os with limited capability of endosomal escape eventually protect mRNA in the cytoplasm to induce sustainable mRNA expression. Higher cytoplasmic stability of pDNA compared to mRNA may shift the limiting step in transfection from cytoplasmic stability to endosomal escape capacity, thereby giving an opposite odd-even effect in transfection efficacy. Endosomal escaping capability and nuclease stability of polyplexes are correlated with the modulated protonation behavior in aminoethylene repeats responding to pH, appealing the substantial importance of chemistry to design polycation structures for promoted mRNA transfection.

Optimized rod length of polyplex micelles for maximizing transfection efficiency and their performance in systemic gene therapy against stroma-rich pancreatic tumors.

Poly(ethylene glycol) (PEG) modification onto a gene delivery carrier for systemic application results in a trade-off between prolonged blood circulation and promoted transfection because high PEG shielding is advantageous in prolonging blood retention, while it is disadvantageous with regard to obtaining efficient transfection owing to hampered cellular uptake. To tackle this challenging issue, the present investigation focused on the structure of polyplex micelles (PMs) obtained from PEG-poly(l-lysine) (PEG-PLys) block copolymers characterized as rod-shaped structures to seek the most appreciable formulation. Comprehensive investigations conducted with particular focus on stability, PEG crowdedness, and rod length, controlled by varying PLys segment length, clarified the effect of these structural features, with particular emphasis on rod length as a critical parameter in promoting cellular uptake. PMs with rod length regulated below the critical threshold length of 200 nm fully exploited the benefits of cross-linking and the cyclic RGD ligand, consequently, exhibiting remarkable transfection efficiency comparable with that of ExGen 500 and Lipofectamine(®) LTX with PLUS™ even though PMs were PEG shielded. The identified PMs exhibited significant antitumor efficacy in systemic treatment of pancreatic adenocarcinoma, whereas PMs with rod length above 200 nm exhibited negligible antitumor efficacy despite a superior blood circulation property, thereby highlighting the significance of controlling the rod length of PMs to promote gene transduction.

Muscle-targeted hydrodynamic gene introduction of insulin-like growth factor-1 using polyplex nanomicelle to treat peripheral nerve injury.

The recovery of neurologic function after peripheral nerve injury often remains incomplete because of the prolonged reinnervation process, which leads to skeletal muscle atrophy and articular contracture from disuse over time. To rescue the skeletal muscle and promote functional recovery, insulin-like growth factor-1 (IGF-1), a potent myogenic factor, was introduced into the muscle by hydrodynamic injection of IGF-1-expressing plasmid DNA using a biocompatible nonviral gene carrier, a polyplex nanomicelle. In a mouse model of sciatic nerve injury, the introduction of IGF-1 into the skeletal muscle of the paralyzed limb effectively alleviated a decrease in muscle weight compared with that in untreated control mice. Histologic analysis of the muscle revealed the IGF-1-expressing plasmid DNA (pDNA) to have a myogenic effect, inducing muscle hypertrophy with the upregulation of the myogenic regulatory factors, myogenin and MyoD. The evaluation of motor function by walking track analysis revealed that the group that received the hydrodynamic injection of IGF-1-expressing pDNA using the polyplex nanomicelle had significantly early recovery of motor function compared with groups receiving negative control pDNA and untreated controls. Early recovery of sensation in the distal area of sciatic nerve injury was also induced by the introduction of IGF-1-expressing pDNA, presumably because of the effect of secreted IGF-1 protein in the vicinity of the injured sciatic nerve exerting a synergistic effect with muscle hypertrophy, inducing a more favorable prognosis. This approach of introducing IGF-1 into skeletal muscle is promising for the treatment of peripheral nerve injury by promoting early motor function recovery.

An injectable spheroid system with genetic modification for cell transplantation therapy.

The new methodology to increase a therapeutic potential of cell transplantation was developed here by the use of three-dimensional spheroids of transplanting cells subsequent to the genetic modification with non-viral DNA vectors, polyplex nanomicelles. Particularly, spheroids in regulated size of 100-µm of primary hepatocytes transfected with luciferase gene were formed on the micropatterned culture plates coated with thermosensitive polymer, and were recovered in the form of injectable liquid suspension simply by cooling the plates. After subcutaneously transplanting these hepatocyte spheroids, efficient transgene expression was observed in host tissue for more than a month, whereas transplantation of a single-cell suspension from a monolayer culture resulted in an only transient expression. The spheroid system contributed to the preservation of innate functions of transplanted hepatocytes in the host tissue, such as albumin expression, thereby possessing high potential for expressing transgene. Intravital observation of transplanted cells showed that those from spheroid cultures had a tendency to localize in the vicinity of blood vessels, making a favorable microenvironment for preserving cell functionality. Furthermore, spheroids transfected with erythropoietin-expressing DNA showed a significantly higher hematopoietic effect than that of cell suspensions from monolayer cultures, demonstrating high potential of this genetically-modified spheroid transplantation system for therapeutic applications.

Targeted gene delivery by polyplex micelles with crowded PEG palisade and cRGD moiety for systemic treatment of pancreatic tumors.

Adequate retention in systemic circulation is the preliminary requirement for systemic gene delivery to afford high bioavailability into the targeted site. Polyplex micelle formulated through self-assembly of oppositely-charged poly(ethylene glycol) (PEG)-polycation block copolymer and plasmid DNA has gained tempting perspective upon its advantageous core-shell architecture, where outer hydrophilic PEG shell offers superior stealth behaviors. Aiming to promote these potential characters toward systemic applications, we strategically introduced hydrophobic cholesteryl moiety at the ω-terminus of block copolymer, anticipating to promote not only the stability of polyplex structure but also the tethered PEG crowdedness. Moreover, Mw of PEG in the PEGylated polyplex micelle was elongated up to 20 kDa for expecting further enhancement in PEG crowdedness. Furthermore, cyclic RGD peptide as ligand molecule to integrin receptors was installed at the distal end of PEG in order for facilitating targeted delivery to the tumor site as well as promoting cellular uptake and intracellular trafficking behaviors. Thus constructed cRGD conjugated polyplex micelle with the elevated PEG shielding was challenged to a modeled intractable pancreatic cancer in mice, achieving potent tumor growth suppression by efficient gene expression of antiangiogenic protein (sFlt-1) at the tumor site.

Multifunctional polyion complex micelle featuring enhanced stability, targetability, and endosome escapability for systemic siRNA delivery to subcutaneous model of lung cancer.

For systemic small interfering RNA (siRNA) delivery to tumor mass, a multifunctional polyion complex micelle was constructed with a block copolymer bearing a targeting ligand and a micelle-stabilizing moiety as well as an endosome-disrupting cationic unit. The block copolymer was comprised of poly(ethylene glycol) (PEG) and a polyaspartamide derivative with flanking cationic tetraethylenepentamine (TEP) moiety (PAsp(TEP)), in which the distal ends of PEG and PAsp(TEP) were further installed with cyclic RGD (cRGD) peptide ligand and cholesteryl (Chol) moiety, respectively. The resulting polymer was confirmed to form siRNA-loaded micelle with a diameter of sub 50 nm and a narrow size distribution. In the stability assays with fluorescently labeled siRNA, the terminal Chol moiety significantly suppressed both the rapid dissociation of the micelles in the serum-containing medium and their rapid elimination from the bloodstream, presumably due to its hydrophobic interactions in the micellar core. Moreover, the targeting cRGD ligand, associated with the stabilizing moiety, significantly enhanced the accumulation of siRNA-loaded micelle in a subcutaneous lung (A549) tumor, compared to a non-targeted control, after systemic administration. Ultimately, significant tumor growth inhibition was achieved by systemic administration of the targeted/stabilized micelle incorporating polo-like kinase 1 (Plk1) siRNA with negligible liver toxicity, consistent with the significant sequence-specific gene silencing of Plk1 in the tumor tissue. These results demonstrated the therapeutic potential of cRGD-PEG-PAsp(TEP)-Chol/siRNA micelle for systemic siRNA delivery toward cancer therapy.

Phenylboronic acid-installed polymeric micelles for targeting sialylated epitopes in solid tumors.

Ligand-mediated targeting of nanocarriers to tumors is an attractive strategy for increasing the efficiency of chemotherapies. Sialylated glycans represent a propitious target as they are broadly overexpressed in tumor cells. Because phenylboronic acid (PBA) can selectively recognize sialic acid (SA), herein, we developed PBA-installed micellar nanocarriers incorporating the parent complex of the anticancer drug oxaliplatin, for targeting sialylated epitopes overexpressed on cancer cells. Following PBA-installation, the micelles showed high affinity for SA, as confirmed by fluorescence spectroscopy even at intratumoral pH conditions, i.e., pH 6.5, improving their cellular recognition and uptake and enhancing their in vitro cytotoxicity against B16F10 murine melanoma cells. In vivo, PBA-installed micelles effectively reduced the growth rate of both orthotopic and lung metastasis models of melanoma, suggesting the potential of PBA-installed nanocarriers for enhanced tumor targeting.