Thermo-responsive targeting of polymeric micelles by controlling the cellular uptake based on the change of their surface arginine density

Yamada, Sota; Sasaki, Eita; Ohno, Hisashi; Nagase, Kenichi; Hanaoka, Kenjiro

doi:10.1038/s42004-025-01707-8

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Published: 04 November 2025

Thermo-responsive targeting of polymeric micelles by controlling the cellular uptake based on the change of their surface arginine density

Communications Chemistry volume 8, Article number: 325 (2025) Cite this article

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Abstract

Stimulus-responsive nanocarriers are good candidates for targeted drug delivery. Herein, inspired by the existence of a clear threshold number of arginine residues in oligoarginines for cell-penetrating peptide (CPP) activity, we developed a strategy to control the CPP activity by changing the local arginine density for thermo-responsive targeting. We constructed polymeric micelles whose shell consists of a thermo-responsive polymer based on N-isopropylacrylamide, with a low density of arginine moieties (named Arg-TRM). At physiological temperature (37 °C), internalization of Arg-TRM into cells was small and comparable to that of micelle without arginine. In contrast, upon heating at 42 °C, the arginine density on the micellar surface was increased by thermo-responsive shrinkage of the shell, thereby switching on the CPP activity and enabling efficient cellular uptake. The response of Arg-TRM at 42 °C occurred within a few minutes and the intracellular uptake was rapidly enhanced from 5 min after the heating. This response was transient, thus enabling reversible control of the enhancement by heating. As proof-of-concept, we show that intravenously administered Arg-TRM was effectively accumulated in one ear of a normal mouse by local heating. These results indicate that Arg-TRM is a promising drug carrier for on-demand targeted drug delivery in response to mild external heating.

Introduction

On-demand delivery of drugs to sites of disease is effective to increase therapeutic efficacy and reduce off-target side effects. Stimulus-responsive nanocarriers responding to various internal stimuli (e.g., pH, redox potential, enzymes) or external stimuli (e.g., magnetic field, light, ultrasound, heat) have been developed for this purpose^1,2. Since internal stimuli rely on the biological environment of the target cells or tissues, external stimuli, which can be applied at any desired time and location, are more suitable for facilitating on-demand delivery. By utilizing external stimuli to modulate the intracellular uptake of a drug carrier, tissue- and cell-specific delivery can be achieved. However, external stimuli often require a long period of application. Thus, the development of drug carriers that are rapidly internalized into the target cells upon specific stimulation is important for clinical applications.

Cell-penetrating peptides (CPPs) are a powerful tool for accelerating the intracellular uptake of drug carriers³. However, CPPs show a universal action regardless of cell type⁴, which leads to nonspecific uptake of drug carriers. Therefore, stimulus-responsive control of CPP activity has been developed to enable the utilization of CPPs for targeted delivery^5,6. So far, this has been achieved by masking/exposure of peptides. Thus, to prevent off-target uptake of drug carriers, the CPPs are initially inactivated by direct binding with shielding domains^{7,8,9,10,11,12,13,14,15,16,17} or by steric hindrance with bulky polymers^{18,19,20,21,22,23}. When required, the CPPs are activated by a stimulus-triggered exposure, enhancing the uptake of the drug carrier into the target cells. In the direct binding approach, CPP activity is effectively blocked with acid-labile^7,8,9,10,11 and enzyme-labile protecting groups¹², or inhibitory domains connected via UV-cleavable¹³, pH-cleavable^14,15, and enzymatically degradable^16,17 linkers. However, since the exposure of the peptides following stimulus-triggered release of the shielding domains is irreversible, there is still a concern about off-target uptake from the systemic circulation. On the other hand, in the steric hindrance approach, reversible control of CPP activity has been achieved by pH-sensitive pop-up of polymers^{18,19,20,21,22} and near-infrared light-induced collapse of polymers²³. However, the conformational changes of the stimulus-responsive polymers have to be large enough to effectively expose the peptide on the surface of the drug carrier, and the response time to achieve sufficient cellular uptake is relatively long. Thus, a superior strategy to enable the CPP activity to be controlled in a reversible, rapid, and efficient manner in response to a stimulus is still required.

Oligoarginine is an artificial CPP that displays a clear threshold for cellular uptake efficacy, depending on the number of arginine units; namely, oligoarginines larger than 6-mer (Arg₆) are dramatically internalized while the 5-mer (Arg₅) and smaller oligoarginines are not^24,25. Interestingly, MacEwan et al. demonstrated that the CPP activity could be induced by stimulus-triggered assembly of diblock polymers containing Arg₅ (i.e., below the threshold by itself)²⁶. In addition, not only linear peptides but also branched arginine-rich peptides showed cell-penetrating ability^27,28. Thus, the local density of the arginine unit appears to be important for the threshold effect of oligoarginines.

Herein, we propose a novel strategy for controlling CPP activity by changing the arginine density to achieve thermo-responsive targeting. Specifically, we hypothesized that intracellular uptake of a drug carrier would be dramatically reduced/enhanced if the local arginine density on the carrier surface is slightly decreased/increased across the threshold. We anticipated that by utilizing such a small change of the local density to turn on the CPP activity, sufficient cellular uptake would be achieved with a short response time. We designed polymeric micelles whose shell consists of a thermo-responsive polymer based on N-isopropylacrylamide (NIPAAm) containing arginine moieties at a low density. Poly(NIPAAm) (PNIPAAm) and its copolymer exhibit a reversible phase transition at the lower critical solution temperature (LCST). Below the LCST, the polymer chains are extended due to interaction with surrounding water molecules, while above the LCST the polymer chains are dehydrated and shrink due to intra- and intermolecular hydrophobic and hydrogen bonding interactions^29,30. Thus, at temperature below the LCST, the arginine density of the micelles is expected to be lower than the threshold for cell internalization (OFF state of the CPP activity). Upon heating at a temperature above the LCST, the surface density of arginine exceeds the threshold due to shrinkage of the thermo-responsive shell, switching the CPP activity to the ON state for efficient cellular uptake (Fig. 1).

**Fig. 1: Schematic illustration of the control of CPP activity by arginine-density change.**

As a proof-of-concept, we designed two types of arginine-containing thermo-responsive micelles (TRMs), ^endArg-TRM, where a single molecule of arginine was introduced at the hydrophilic end of the polymer, and ^intraArg-TRM, where multiple arginine units were introduced in the hydrophilic domain of the polymer along with TRM as a control (Fig. 2). In both cases, the density of the arginine moieties is expected to be increased when the thermo-responsive shell shrinks. We examined the thermo-responsiveness of Arg-TRMs and evaluated the enhancement of cell internalization upon heating. In addition, the mechanism of the intracellular uptake was examined by competitive inhibition with arginine and also by using endocytosis inhibitors. Finally, thermo-responsive targeting of systemically injected Arg-TRMs in mice in response to local heating was demonstrated.

**Fig. 2: Molecular design of thermo-responsive micelles (TRMs).**

Results and discussion

Synthesis and characterization of the polymeric micelles

To construct the designed micelles, amphiphilic diblock copolymers were synthesized by two-step reversible addition-fragmentation chain transfer (RAFT) polymerization. To synthesize ^endArg-TRM, acrylamide (AAm) and NIPAAm were copolymerized first to make a hydrophilic shell domain. AAm was copolymerized with NIPAAm to tune the LCST of PNIPAAm to a slightly higher temperature than the physiological temperature²⁹. The resulting P(NIPAAm-co-AAm) was used as a macro-chain transfer agent for the second polymerization step to add a hydrophobic domain consisting of poly(n-butyl methacrylate) (PBMA). Finally, an arginine molecule was conjugated to the hydrophilic end of the amphipathic diblock copolymer, yielding Arg-P(NIPAAm-co-AAm)-b-PBMA (Scheme S1). To obtain ^intraArg-TRM, a similar diblock copolymer was synthesized using the same RAFT polymerization procedure, except that AAm was substituted with an arginine derivative monomer (ArgAAm) in the first polymerization step (Scheme S2). The introduction of arginine moieties into the polymers was confirmed by ¹H NMR, and the copolymerization ratio of ArgAAm in P(NIPAAm-co-ArgAAm) was calculated from the integral intensity of ArgAAm at 4.1 ppm to that of NIPAAm at 3.8 ppm (Supplementary Information). The copolymer compositions, molecular weights, and dispersity of the prepared polymers are shown in Table S1. The average molecular weights of Arg-P(NIPAAm-co-AAm)-b-PBMA and P(NIPAAm-co-ArgAAm)-b-PBMA were 16 kDa and 20 kDa, respectively, with relatively low polydispersities (1.1–1.3). The percentage of hydrophilic domains in the amphiphilic diblock copolymers was higher (70–80%) than the value commonly considered necessary to form spherical micelles (>45%)³¹.

The ^endArg-TRM, ^intraArg-TRM, and a control TRM without arginine were prepared from Arg-P(NIPAAm-co-AAm)-b-PBMA, P(NIPAAm-co-ArgAAm)-b-PBMA, and P(NIPAAm-co-AAm)-b-PBMA, respectively, by slowly adding the polymer solution in N,N-dimethylacetamide to water, followed by dialysis. A fluorescent hydrophobic indocarbocyanine dye (DiD or DiO) was encapsulated in the hydrophobic cores of the micelles. Micelles loaded with DiD (a red fluorescent dye) were used to evaluate micellar uptake into cells and accumulation in vivo, while DiO (a green fluorescent dye) was encapsulated to analyze size and zeta potential due to its optical compatibility with the measuring instruments. The size, determined by dynamic light scattering (DLS), and the zeta potential of the three TRMs are shown in Table 1. The micelle sizes of Arg-TRMs and TRM were different, presumably due to the introduction of the arginine moieties. When TRM-2, an analog of ^endArg-TRM, was prepared from an amphipathic diblock copolymer without the arginine moiety, which is the precursor of Arg-P(NIPAAm-co-AAm)-b-PBMA (Scheme S1), the size of TRM-2 was larger (80 ± 0.3 nm) than that of ^endArg-TRM (54 ± 0.2 nm shown in Table 1), even though these polymers are the same except for the conjugation of an arginine moiety to the polymer terminal. Since salt concentration had no significant effect on the micelle sizes (Table S2), the most likely explanation seems to be that introduction of the arginine moiety changes the inter-polymer interaction during micelle formation of Arg-TRMs.

Table 1 Characterization of the polymeric micelles

Full size table

It was reported that polymeric micelles containing PNIPAAm shells exhibit a phase separation upon heating^30,32. To evaluate micellar phase transition, the cloud-point temperature (T_cp) was measured based on the change in transmittance. In our design, the T_cp of the micelles was tuned to between 39 and 42 °C by adjusting the composition of NIPAAm and hydrophilic co-monomer, AAm or ArgAAm (Fig. 3A and Table 1). Upon heating at 42 °C (higher than the T_cp), the optical transmittance of the micelles was dramatically decreased within a few minutes (Fig. 3B). In contrast, no change was observed at 37 °C (lower than the T_cp) even after 30 min. We confirmed that Arg-TRMs responded quickly and specifically to heating at a temperature above the T_cp. We also investigated the thermo-responsive behavior of the micelles by measuring the temperature dependence of their hydrodynamic diameter, because micellar aggregation could occur above the T_cp due to increased hydrophobic interaction between PNIPAAm-shell micelles^33,34. The hydrodynamic diameter of the micelles was clearly increased at temperature above the T_cp even for ^endArg-TRM and ^intraArg-TRM containing additional hydrophilic arginine moieties (Fig. 3C). Furthermore, the observed aggregation upon heating at 42 °C completely disappeared after cooling at 37 °C, revealing that the Arg-TRMs responded reversibly to the temperature change (Fig. 3D).

**Fig. 3: Thermo-responsiveness of the polymeric micelles.**

Next, the effect of heating on the surface charge of the micelles was investigated. The zeta potentials of ^endArg-TRM and ^intraArg-TRM were increased when the temperature was raised above the micellar T_cp, from 3–4 mV at 37 °C to 10 mV at 42 °C (Fig. 3E). The observed positive charge was most likely derived from the guanidine moiety of arginine, because the oligoarginine-modified nanoparticles showed a positive correlation between the zeta potential and the number of modified peptides^35,36,37. This would be consistent with the increase of the opposite charge at 42 °C for TRM, which possesses negatively charged carboxyl groups on the surface. (Fig. 3E). These results support the occurrence of thermo-responsive condensation of arginine (increase of arginine density) on the surface of ^endArg-TRM and ^intraArg-TRM upon heating.

Thermo-responsive cellular uptake of polymeric micelles based on increase of surface arginine density

To test the thermo-responsive cellular uptake of the polymeric micelles, Arg-TRMs encapsulating a fluorescent dye, DiD, were applied to murine colon carcinoma (Colon-26) cells. We confirmed that incubation at 42 °C within 1 h was not toxic to the cells (Fig. S1), similar to previously reported heat tolerance studies in various cell lines³⁸. The cells were incubated with the micelles, ^endArg-TRM and ^intraArg-TRM, as well as TRM (non-arginine control), for 30 min at 42 °C. We also checked that these heating conditions did not affect the amount of DiD contained in the micelles (Fig. S2) or the cell membrane permeability by measuring the intracellular retention of Calcein, a membrane-impermeable dye (Fig. S3). As expected, the cellular uptake levels of ^endArg-TRM and ^intraArg-TRM were significantly increased at 42 °C compared to those at 37 °C (Fig. 4A), showing that Arg-TRMs uptake is enhanced by heating above the T_cp. Since the uptake levels at 37 °C were similar between Arg-TRMs and TRM, it was confirmed that the enhanced uptake of Arg-TRMs was not simply due to the introduced arginine. Although it was assumed that non-specific uptake would be more likely to occur with Arg-TRMs than TRM due to the smaller size^39,40, the intracellular uptake at 37 °C did not correlate with the micellar size. Since these micelles are within the diameter range of typical endosomes^41,42, the size difference may not affect the cellular uptake at 37 °C. The fold increases in cellular uptake at 42 °C versus 37 °C were calculated to predict the delivery efficiency to the target tissue upon heating compared to off-target tissues at physiological temperature. ^endArg-TRM, ^intraArg-TRM and TRM showed fold increases at 1.9, 4.3, and 1.1, respectively. These results demonstrated that the thermo-responsive uptake of the micelles could be enhanced by the introduction of the arginine moieties, and the enhancement was much greater for ^intraArg-TRM than for ^endArg-TRM. We confirmed that the cytotoxicity of ^intraArg-TRM at 37 °C was negligible over a wide range of the micellar concentration (10–3000 µg/mL) (Fig. S4). Therefore, we decided to utilize ^intraArg-TRM for the following experiments.

**Fig. 4: Intracellular uptake of the arginine-containing thermo-responsive micelles.**

To further examine the effect of the phase transition and the number of arginine units per polymer on the cellular uptake, two additional ^intraArg-type micelles were prepared as controls. The first, ^intraArg-TRM(high), was prepared to display a higher T_cp (49.0 °C) than 42 °C due to the presence of additional AAm in the polymer (Fig. S5 and Table S3), while the molar ratio of arginine units was maintained almost the same as that of ^intraArg-TRM (Table S1). Unlike ^intraArg-TRM, the zeta potential of ^intraArg-TRM(high) was not significantly increased at 42 °C compared with that at 37 °C (Fig. S6). The cellular uptake level of ^intraArg-TRM(high) was comparable to that of ^intraArg-TRM at 37 °C, but was not increased at 42 °C (Fig. 4B). This result indicates that the phase transition above the LCST was essential to enhance the ^intraArg-TRM uptake. The second control micelle, ^intraArg-TRM(many), was designed to possess a 1.5-fold increased number of arginine units compared to that of ^intraArg-TRM while keeping the T_cp (41.8 °C) lower than 42 °C (Tables S1, S3 and Fig. S5). The zeta potential of ^intraArg-TRM(many) at 37 °C (below the T_cp) was 9 mV (Figure S6), which was comparable to that of ^intraArg-TRM at 42 °C (above the T_cp) (Fig. 3E). At 37 °C (lower than the T_cp), the cellular uptake level of ^intraArg-TRM(many) was 4-fold higher than that of ^intraArg-TRM (Fig. 4B). The reason for this increased uptake of ^intraArg-TRM(many) is probably that the arginine density on the micellar surface exceeded the threshold for cellular internalization even without shrinking of the polymer. Further, the zeta potential and the cellular uptake level of ^intraArg-TRM(many) were increased by heating at 42 °C (higher than the T_cp) (Figs. 4B and S6), which is consistent with a previous report that the cellular uptake level of oligoarginines longer than Arg₆ increases as the number of arginine units is increased²⁴. These results support the conclusion that the enhancement of the cellular uptake of ^intraArg-TRM upon heating was induced by an increment of the local arginine density due to the phase transition.

Next, three polymeric micelles possessing fewer arginine units than ^intraArg-TRM were prepared from mixed polymer solutions of P(NIPAAm-co-ArgAAm)-b-PBMA and P(NIPAAm-co-AAm)-b-PBMA (Fig. 4C and Table S4). The T_cp of these mixed micelles was between 40.0 and 41.3 °C (Fig. S7). In addition to comparing TRM and Arg-TRMs, we found that the sizes of the mixed micelles decreased as the ratio of the arginine units was increased (Fig. S8), and the cellular uptake at 37 °C was not affected by the micellar size (Fig. S9). The cellular uptake of only ^intraArg-TRM, but not the other micelles, was sharply increased at 42 °C (Fig. 4D), supporting our hypothesis that the arginine density threshold is critical for the cellular uptake. It is interesting that the enhancement of the cellular uptake of ^endArg-TRM is greater than that of the three mixed micelles (Fig. 4A, D) even though the total number of arginine moieties was estimated to be higher for the latter micelles (Table S1). These results suggest that there is no simple correlation between the level of cellular uptake and the number of arginine units in the micelles. In the mixed micelles and ^intraArg-TRM, only the arginine moieties near the micellar surface might contribute to the cellular uptake, while the arginine moiety of ^endArg-TRM might interact more efficiently with the cells because it is exposed at the surface.

Mechanism and pathway of cellular uptake of ^intraArg-TRM

Since extracellular arginine molecules are known to be taken up into cells via cationic amino acid transporters^43,44, we investigated whether or not these transporters were involved in the micellar uptake. ^intraArg-TRM was added to the cells in culture medium supplemented with arginine hydrochloride within the concentration range where the calculated osmolality does not exceed the physiological range. The presence of the excess arginine did not significantly decrease the cellular uptake of ^intraArg-TRM at 42 °C (Fig. 5A). Thus, cationic amino acid transporters are unlikely to be involved in the cellular uptake of ^intraArg-TRM.

**Fig. 5: Mechanisms of cellular uptake of ^intraArg-TRM.**

When we observed the intracellular distribution of ^intraArg-TRM loaded with DiD by confocal fluorescence microscopy, the fluorescence dots representing the dye were localized inside the cells (Fig. 5B). Co-staining with a lysosome-specific dye clearly showed that ^intraArg-TRM was localized in lysosomes, suggesting that it is internalized at 42 °C via endocytosis. Therefore, we next used various endocytosis inhibitors^45,46 to examine the endocytic pathway of ^intraArg-TRM at 42 °C (Fig. 5C). No decrease of the uptake was observed in the presence of clathrin-mediated endocytosis inhibitors (chlorpromazine and dansylcadaverine) or a caveolae-mediated endocytosis inhibitor (nystatin). An unexpected increase in the uptake was observed with filipin III, a caveolae-mediated endocytosis inhibitor, but this might be due to permeabilization of the cell membrane⁴⁷. On the other hand, the uptake was significantly suppressed by a lipid raft-mediated endocytosis inhibitor (methyl-β-cyclodextrin) and by macropinocytosis inhibitors (EIPA, cytochalasin D and wortmannin). Moreover, cellular uptake of 70 kDa dextran, a marker molecule of macropinocytosis^45,46, was increased by treatment with ^intraArg-TRM at 42 °C compared with that at 37 °C. In contrast, no increase of the dextran uptake was observed upon co-incubation with TRM at 42 °C (Fig. 5D). These results indicate that macropinocytosis was involved in the ^intraArg-TRM uptake at 42 °C. This is consistent with findings for oligoarginine-modified nanoparticles^{35,36,48,49,50}, and supports the idea that the CPP activity of ^intraArg-TRM was turned on by heating.

We considered two possible pathways for the cellular internalization of ^intraArg-TRM (Fig. 5E). Upon heating, the shrinkage of the thermo-responsive polymer reduces the volume of the micellar shell and increases the surface density of the micelles. In the first pathway, the structural change of the micellar shell simply increases the local density of the arginine moieties above the threshold value for cellular uptake, leading to the internalization of the shrunken micelles. In the second pathway, aggregation of the heated micelles via hydrophobic interactions further increases the arginine density, leading to internalization as micellar aggregates.

To observe these thermo-responsive changes more precisely, we tuned the phase transition of ^intraArg-TRM to occur over a wider temperature range, based on the effect of salt concentration on the phase transition of PNIPAAm⁵¹. By using a low-salt-containing buffer including 10 mM NaCl (HEPES-NaCl) instead of PBS containing 155 mM NaCl, the T_cp of ^intraArg-TRM was increased to 49.6 °C, and the phase transition occurred over an 8-degree temperature range (Fig. S10). When ^intraArg-TRM was heated in HEPES-NaCl at a temperature slightly lower than the T_cp (48 °C), an increase of the zeta potential preceded the increase of the hydrodynamic diameter and reached a plateau in 10 min, while the hydrodynamic diameter continued to increase for over 30 min (Fig. S11). These results indicate that the local density of arginine was increased by shrinkage rather than by aggregation. Thus, cellular uptake via the first pathway may be induced in the early stage of the heat activation (Fig. 5E). However, ^intraArg-TRM quickly aggregated in a few minutes of heating, both in PBS (Fig. S12) and in the culture medium containing 10% FBS (Fig. S13). The intracellular uptake of ^intraArg-TRM at 42 °C was still efficient even when the micellar aggregates prepared by pre-heating in the medium were incubated with the cells (Fig. S14). Furthermore, another experiment, in which various concentrations of ^intraArg-TRM were tested, showed that aggregate formation and intracellular uptake at 42 °C were correlated to some extent (Figs. S12 and S15). Therefore, it is likely that ^intraArg-TRM is internalized into the cells mainly in the form of micellar aggregates in the middle-to-late stage of the heat activation, according to the second pathway (Fig. 5E).

Reversible and rapid activation of cellular uptake of ^intraArg-TRM

To achieve stimulus-responsive drug delivery, precise control of uptake in response to specific stimulation is desirable. Thus, we investigated the reversibility of the CPP activity in response to temperature change across the T_cp (39.6 °C of ^intraArg-TRM). During cycles of temperature change between 37 °C (physiological temperature) and 42 °C, the intracellular uptake of the micelles proceeded well at 42 °C, whereas only slow uptake was observed at 37 °C (Fig. 6A). This result shows that the enhancement of the intracellular uptake of ^intraArg-TRM by heating was transient. The reversibility of the enhancement would enable precise control of drug delivery to the target cells.

**Fig. 6: Thermo-responsive cellular uptake of ^intraArg-TRM.**

In addition to reversibility, rapid internalization into the target cells upon stimulation is important for on-demand delivery of a drug carrier. Although the standard duration for clinical hyperthermia treatment is within 1 h, conventional drug carriers comprised of thermo-responsive polymers often required heating durations of several hours or more to provide significant cellular uptake^{33,52,53,54,55,56}. Thus, we investigated the relationship between duration of heating and the level of intracellular uptake of ^intraArg-TRM. To our delight, initiation of cellular uptake of ^intraArg-TRM was already observable within just 5 min after the start of heating, and the fold increase of the uptake exceeded 4-fold after heating for 15 min (Fig. 6B). This fold increase in response to 15 min of stimulation is estimated to be higher than previously developed methods for the reversible control of CPP activity^{18,19,20,21,22,23}. These results demonstrate that intracellular uptake of ^intraArg-TRM can be induced both quickly and effectively in response to heating.

Additionally, the intracellular uptake of ^intraArg-TRM continued to increase with heating for longer than 30 min (Fig. 6B), while ^intraArg-TRM (57 nm in diameter) formed aggregates with an apparent particle size of 1 µm on heating at 42 °C for 10−15 min in the medium (Fig S13). These results support the idea that ^intraArg-TRM can be internalized into the cells as aggregates. However, particle size generally affects cellular uptake, for example, being slower for larger particles^39,40. Importantly, the uptake of TRM (non-arginine control) did not increase even after 60 min of heating (Fig. S16), which is similar to the previously reported micelles consisting of the thermo-responsive shell based on NIPAAm^33,51,52. This is probably because the size of the micelle aggregates exceeds that of endosomes mediating clathrin- and caveolae-dependent endocytosis, which typically involved in the uptake of nanoparticles and have a diameter of 85–150 nm^41,42. In contrast, macropinosomes are much larger, with a diameter of up to 5 μm^57,58. Thus, macropinosomes might mediate the uptake of ^intraArg-TRM aggregates. We considered that the CPP activity of oligoarginine, which induce macropinocytosis, contributes to ^intraArg-TRM overcoming the unfavorable effect of the increased size of the thermo-responsive carrier.

Thermo-responsive targeting of ^intraArg-TRM in vivo

Based on the reversible and rapid intracellular uptake of the ^intraArg-TRM in response to the temperature change, we then examined on-demand delivery of the micelles in vivo. We used a dose of 1.0 mg polymer per mouse because the expected micelle concentration in the blood (10% of body weight) under this condition is the same as used in the standard cellular uptake experiments described above (400 µg/mL micelle concentration). When we injected ^intraArg-TRM at this dose, the mice remained apparently healthy over 3 weeks and showed no body weight loss (Fig. S17), suggesting that ^intraArg-TRM exhibits good biocompatibility as a drug carrier.

Next, DiD-loaded ^intraArg-TRM was intravenously administered to the mice, and only the left ears of each mouse were heated for 30 min at 42 °C. This procedure is shorter than the standard duration and a feasible temperature for clinical hyperthermia treatment^59,60,61. Upon heating, the blood vessels in the left ear became dilated (Fig. S18), but there was no apparent difference between the two ears after 24 h (Fig. S19), suggesting that the treatment was not invasive. We then evaluated the accumulation of ^intraArg-TRM in the left ear in response to local heating. In vivo images showed that the fluorescence signals of DiD from the left ear (heated) were clearly stronger than those from the right ear (unheated) (Fig. 7A), and this enhancement was maintained at 24 h after the micelle injection. To evaluate the difference quantitatively, we measured the accumulation rate of ^intraArg-TRM in the ear on each side by extracting DiD of ^intraArg-TRM from the excised ears. As expected, the median value (interquartile range) was significantly increased from 0.19% of injected dose (0.02–0.42) for the right ear (unheated) to 0.88% of injected dose (0.80–0.94) for the left ear (heated) (Fig. 7B). The fold increase of the median value for the left ear versus the right ear was calculated as 4.7. In contrast, the corresponding fold increases of TRM (without arginine) and ^intraArg-TRM(high) (T_cp 49.0 °C) were 1.2 and 1.1, respectively. These results confirm that both the arginine moieties and micellar phase transition at a temperature above LCST are important for the thermo-responsive targeting of ^intraArg-TRM in vivo, in accordance with the findings in the cellular experiments. Furthermore, we observed the localization of ^intraArg-TRM in the left ear (heated) by confocal fluorescence microscopy of frozen sections. The fluorescence signals of DiD were mainly observed inside the cell and were scarcely observed in the extracellular matrix, where no cell nuclei are present (Fig. 7C), indicating that ^intraArg-TRM had been internalized into cells in the heated tissues. Immunostaining for CD31 (a marker of endothelial cells) further showed that the intracellular uptake of ^intraArg-TRM occurred not only around blood vessels, but also in areas distinct from the vessels. These results suggest that the micelles diffused in the tissue and responded to the heating, turning on the CPP activity. Based on reports that local tissue heating at mild high temperatures (between 39 and 42 °C) promotes the vascular permeability in tumors^62,63,64, it can be occurred that ^intraArg-TRM, which is smaller than the heating-expanded endothelial gaps, penetrates from the blood vessels into the surrounding tissues.

**Fig. 7: In vivo targeting of ^intraArg-TRM to mouse ears.**

Conclusions

We have developed a strategy for controlling the CPP activity of drug carriers by changing the local arginine density. To the best of our knowledge, this is the first time that stimulus-responsive control of CPP activity has been achieved without the use of peptides. Our results demonstrate that the density of arginine is indeed important for the CPP activity of oligoarginines. The designed micelle ^intraArg-TRM, which consists of a PNIPAAm-based thermo-responsive polymer with multiple arginine moieties in the hydrophilic domain, responded specifically to temperature above the LCST, which induced a phase change that turned on the cell internalization activity. The activation relies on shrinkage of the micellar shell at temperature above the LCST with forming aggregates, resulting in an increase of the local arginine density on the surface of the micelles. The control micelle without arginine (TRM) did not exhibit such cell internalization activity, although it responded similarly to heating, becoming more hydrophobic on the surface and forming aggregates, thereby highlighting the importance of the arginine moieties for the observed cellular uptake of ^intraArg-TRM. Intriguingly, a clear threshold of arginine density on the surface of the nanocarriers exists for the cellular uptake, as has been observed for oligoarginine peptides^24,25. This was established by the experiments using control micelles with an increased number of arginine units (^intraArg-TRM(many)), and three mixed micelles with reduced ratios of arginine-containing polymers. Since arginine moieties near the surface likely contribute to the internalization activity, as found for oligoarginine-modified micelles⁶⁵, placing the arginine moieties at appropriate positions in the nanocarriers is considered to be a key factor in our design. We also demonstrated that the enhancement of ^intraArg-TRM uptake is reversible in response to temperature change across the LCST. Considering the rapid and efficient internalizing ability of ^intraArg-TRM upon heating, it appears to be a promising candidate for use as an on-demand drug delivery system. As a proof-of-concept, we applied ^intraArg-TRM to mice and achieved thermo-responsive targeting of the micelles to a specific location, an ear. The micelles were efficiently distributed in the heated tissues, and this selective targeting is a promising feature for potential applications in cancer therapy. Notably, our system required local heating at 42 °C for only 30 min, and intravenously administered ^intraArg-TRM in mice showed no apparent toxicity. Although thermal damage to target tissues should be considered as effects of heating⁶⁶, several studies have shown no serious damage to heated normal tissues by hyperthermia in animal models^67,68,69 and clinical trials⁷⁰. Thus, Arg-TRM appears to have clinical potential for on-demand drug delivery in response to external heating.

Method

Materials

NIPAAm was kindly provided by KJ Chemicals (Tokyo, Japan) and purified by recrystallization from n-hexane. AAm was purchased from Nacalai Tesque (Kyoto, Japan) and used without further purification. 4-Cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl] pentanoic acid (CDTPA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). V-501 and butyl methacrylate (BMA) were purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan), and BMA was passed through an inhibitor removers packed column (Sigma-Aldrich) before use. Arginine derivative of acrylamide (ArgAAm) was synthesized according to a previously published procedure⁷¹. H-Arg(Pbf)-O^tBu･HCl (arginine protected with a 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl group (Pbf) and a tert-butyl group (^tBu)) was purchased from Watanabe Chemical Industries (Hiroshima, Japan). DMT-MM was purchased from Tokyo Chemical Industries (Tokyo, Japan). Fluorescent dye, 1,1’-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) was obtained from Setareh Biotech (Eugene, OR, USA) and 3,3′-dioctadecyloxacarbo-cyanine perchlorate (DiO) was obtained from Takara Bio (Shiga, Japan). Dialysis membranes (Spectra/Por 7, molecular weight cut-off (MWCO) 1 and 15 kDa) were purchased from Spectrum Laboratories (Rancho Dominguez, CA, USA). Hydrophilic syringe filter (Minisart RC25) was obtained from Sartorius (Goettingen, Germany). Other organic solvents and chemical reagents were purchased from Fujifilm Wako Pure Chemical Corporation. RPMI-1640 medium, Dulbecco’s phosphate-buffered saline without calcium and magnesium (PBS), LysoTracker-Red and Hanks’ balanced salt solution with calcium and magnesium (HBSS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was obtained from Biosera (Nuaille, France). HEPES and Calcein-AM were obtained from Dojindo Laboratories (Kumamoto, Japan). Accutase was obtained from Nacalai Tesque. Lysis solution (Luciferase Cell Culture Lysis Reagent) was obtained from Promega (Madison, WI, USA). Eight well chamber slides were obtained from WATSON (Tokyo, Japan). Mounting medium (VectaShield) was obtained from Vector Laboratories (Burlingame, CA, USA). Antibiotics, trypsin-EDTA, sucrose and chlorpromazine were obtained from Fujifilm Wako Pure Chemical Corporation. Other endocytosis inhibitors and TRITC-dextran 70 kDa were obtained from Sigma-Aldrich.

Synthesis of Arg-P(NIPAAm-co-AAm)-b-PBMA (^endArg-TRM)

Carboxyl-terminated amphiphilic diblock polymer was prepared by two-step reversible addition−fragmentation chain transfer (RAFT) polymerization. NIPAAm, AAm, and CDTPA as a chain transfer agent (CTA; 156 mg, 0.385 mmol) were dissolved in 1,4-dioxane (15.4 mL) in a two-necked flask. Then, V-501 (10.8 mg, 0.0385 mmol, 0.1 eq.) as a radical initiator was added to the solution. Initial total monomer concentration was 3 M (46.3 mmol, 120 eq., NIPAAm/AAm = 88/12 mol%). The reaction mixture was degassed by bubbling with argon for 30 min, and stirred for 16 h at 74 °C. Then, the reaction solution was diluted with acetone and poured into diethyl ether (150 mL) to precipitate the polymer, which was collected and dried under vacuum to afford P(NIPAAm-co-AAm) as a pale yellow solid (5040 mg, 98%). In the second block preparation, P(NIPAAm-co-AAm) (1500 mg, 0.115 mmol) as the macro-CTA, BMA (908 µL, 5.73 mmol, 50 eq.), and V-501 (6.4 mg, 0.023 mmol, 0.2 eq.) were reacted in 1,4-dioxane (22.9 mL) for 24 h at 74 °C, and then the reaction solution was dialyzed against methanol using dialysis membranes (MWCO 1 kDa) at room temperature for 3 days. The resulting solution was evaporated to dryness to give P(NIPAAm-co-AAm)-b-PBMA as a pale yellow powder (1918 mg, 46%). Next, an arginine moiety was introduced via amide condensation at the carboxyl terminal of the diblock polymer. To a solution of P(NIPAAm-co-AAm)-b-PBMA (300 mg, 0.0186 mmol) in tetrahydrofuran (4.5 mL) was added H-Arg(Pbf)-OtBu･HCl (19.3 mg, 0.0373 mmol, 2.0 eq.) and triethylamine (5.0 eq.). The mixture was cooled to 4 °C, and DMT-MM (15.5 mg, 0.0559 mmol, 3.0 eq.) was added as a powder. This mixture was stirred for 16 h. After further reaction for 16 h at room temperature, the solvent was evaporated and the resulting residue was dissolved in dichloromethane (4.5 mL), followed by a deprotection step. To the solution was added water (0.45 mL) followed by dropwise addition of dichloromethane (CH₂Cl₂)/trifluoroacetic acid (TFA) (v/v = 50/50, 9.0 mL) at 4 °C. The final volume ratio of the solution was CH₂Cl₂/TFA/water = 20/10/1. After reaction for 3 h at room temperature, the polymer was purified using the same method as described for the second block preparation to give Arg-P(NIPAAm-co-AAm)-b-PBMA as a pale yellow powder (289 mg, 95%).

Synthesis of P(NIPAAm-co-ArgAAm)-b-PBMA (^intraArg-TRM)

P(NIPAAm-co-ArgAAm)-b-PBMA was prepared by two-step RAFT polymerization as described above. A mixture of NIPAAm, ArgAAm, CDTPA (33.7 mg, 0.0834 mmol), and V-501 (4.68 mg, 0.0167 mmol, 0.2 eq.) in 1,4-dioxane/DMSO (v/v = 8/2, 8.3 mL) was reacted for 24 h at 74 °C. Initial total monomer concentration was 2 M (16.7 mmol, 200 eq., NIPAAm/ArgAAm = 90/10 mol%). After precipitation in diethyl ether (160 mL), P(NIPAAm-co-ArgAAm) was collected as a pale yellow solid (1247 mg, 58%). In the second block preparation, P(NIPAAm-co-ArgAAm) (300 mg, 0.0214 mmol) was reacted with BMA (238 µL, 1.50 mmol, 70 eq.) and V-501 (1.2 mg, 0.0043 mmol, 0.2 eq.) in DMSO (8.0 mL) for 24 h at 74 °C. The reaction solution was purified by dialysis against methanol to give P(NIPAAm-co-ArgAAm)-b-PBMA as a pale yellow powder (332 mg, 15%).

Characterization of the polymers

¹H NMR spectra were recorded at 400 MHz using a 400-MR spectrometer (Agilent, Santa Clara, CA, USA). The molecular weight of polymers was determined by gel permeation chromatography (GPC) (GPC-8020 system, TOSOH, Tokyo, Japan) using a TSKgel guard column α and TSKgel column α-M with N,N-dimethylformamide containing 10 mM LiCl as a mobile phase. Calibration was done with polyethylene oxide standards.

Preparation of the polymeric micelles

Fifteen milligrams of diblock copolymer was dissolved in 2.0 mL N,N-dimethylacetamide (DMAc) containing fluorescent dye (90 µg of DiD or 30 µg of DiO) and the solution was stirred overnight at room temperature. To 6.0 mL of deionized water stirred at 750 rpm, and the polymer solution was slowly added using a syringe pump at 0.1 mL/min. The resulting solution was dialyzed against deionized water using dialysis membranes (MWCO 15 kDa) at room temperature (20−25 °C) for 1 day. After removal of unloaded dye through 0.45 µm pore syringe filter, the micelle solutions were stored at 4 °C until use.

Characterization of the polymeric micelles

The optical transmittance of the DiD-loaded micelles in PBS was measured at 750 nm using a UV–Vis spectrophotometer V-630 (JASCO, Tokyo, Japan) and a water circulation bath CTU-100 (JASCO) at a heating rate of 1.0 °C/min. The cloud-point temperature (T_cp) of the micelle solution was defined as the temperature showing a 50% decrease in optical transmittance. The hydrodynamic diameters and their distribution of DiO-loaded micelles were measured at 25 °C by DLS using a Zetasizer Nano-ZS (Malvern Instrument, Worcestershire, UK) equipped with a 633 nm laser at a scattering angle of 173°. For evaluation of the reversible response to heating, the hydrodynamic diameters were measured 5 min after reaching the target temperature. The zeta potentials of the DiO-loaded micelles in PBS (pH 7.4) were measured at the indicated temperature using a ELSZ-2KOP zeta potential analyzer (Otsuka Electronics Co., Ltd., Osaka, Japan). For evaluation of the temporal changes of zeta potential in 10 mM HEPES buffer containing 10 mM NaCl (HEPES-NaCl) (pH 7.4), a Zetasizer Nano-ZS was used. The critical micelle concentration (CMC) of the micelles was determined using pyrene as a fluorescence probe. Pyrene solution in acetone (120 µM, 7.5 µL) was added to micelles without fluorescent dye in PBS (1500 µL) at various concentrations (1−1000 µg/mL) and the mixtures were incubated at 20 °C for 16 h before measurements. The fluorescence spectra were recorded (excitation: 338 nm, emission: 350−450 nm) and the CMC value was estimated by extrapolating the crossing-point of the intensity ratio I₃₇₅/ I₃₈₆. The loading amount of DiD in the micelles was calculated from the fluorescence intensity of DiD, measured using an F-7000 fluorescence spectrophotometer (Hitachi High-Tech, Tokyo, Japan) (excitation: 650 nm, emission: 672 nm).

Cell culture

Murine colon carcinoma (Colon-26; RCB2657) cells were obtained from RIKEN Cell Bank (Ibaraki, Japan). Colon-26 cells were cultured on non-coated dishes in RPMI-1640 supplemented 10% FBS, 100 units/mL penicillin G, and 100 mg/L streptomycin. Cells were grown in a CO₂ incubator (humidified atmosphere of 5% CO₂ in air at 37 °C) and subcultured twice a week after washing with PBS and detaching with 0.25% trypsin-EDTA.

Cellular uptake of DiD-loaded micelles

Colon-26 cells were seeded into 24-well plates at 5 × 10⁴ cells/well and allowed to attach for 24 h at 37 °C. Prior to treatment with the micelles, the cells were fluorescently labeled with 200 nM Calcein-AM in RPMI1640 (300 µL/well) at 37 °C for 15 min. DiD-loaded micelles in the medium containing 10% FBS and 13 mM HEPES were added to the cells (300 µL/well). After 30 min at 37 °C or 42 °C in the CO₂ incubator, the cells were washed twice with ice-cold PBS, dissociated with Accutase (100 µL/well) for 10 min, and lysed with 2× lysis solution (100 µL/well) for 15 min in the well plate. After mixing with DMSO (800 µL/well) at 500 rpm on orbital shaker, 200 µL of the resulting mixture was transferred to 96-well black plate and the fluorescence intensity was measured using a Nivo plate reader (PerkinElmer, Waltham, MA, USA) for DiD detection (excitation filter: 600/10 nm, emission filter: 640/30 nm, dichroic mirror: D565, measurement time: 1000 ms) and Calcein detection (excitation filter: 480/30 nm, emission filter: 530/30 nm, dichroic mirror: D500, measurement time: 200 ms). Standard samples were prepared for each micelle to estimate the value for 20% uptake: lysis solution containing each micelle was added to unused colon-26 wells. The fluorescence intensity of Calcein was used to estimate cell number, and the cellular uptake of DiD was normalized by the relative cell number with respect to nontreated cells.

Cell viability assay

Colon-26 cells were seeded into a 96-well plate (black/ clear bottom) at 8 × 10³ cells/well and allowed to attach for 24 h at 37 °C. To evaluate the cytotoxicity of micelles, the medium was replaced with the medium containing micelles (100 µL/well) and the cells were incubated for 24 h in the CO₂ incubator. Then, Cell Counting Kit-8 reagent was added (10 µL/well) and the cells were incubated for a further 1 h. The absorbance of the wells was measured using a TECAN Infinite M1000 plate reader at 450/10 nm. The relative percentage of cell survival was calculated based on the absorbance obtained from a non-treated group taken as 100%. The cytotoxicity of the endocytosis inhibitors was similarly evaluated, except for the incubation periods: the cells were incubated with inhibitors for 3 h, then incubated for 2 h after addition of the assay reagent.

Confocal microscopy

Colon-26 cells were seeded into an 8-well chamber slide at 1.75 × 10⁴ cells/well and allowed to attach for 24 h at 37 °C. Then, the cells were incubated at 42 °C for 30 min with DiD-loaded micelles (200 µL/well). After replacement of the medium with new medium without micelles, the cells were further incubated at 37 °C for 210 min and then incubated with 75 nM LysoTracker-Red for 30 min. The slides were washed with ice-cold HBSS, immersed in 4% paraformaldehyde, and sealed with a mounting medium. Fluorescence images were acquired using an FV3000 confocal fluorescence microscope (Olympus, Tokyo, Japan); 60×, LysoTracker-Red (laser wavelength: 561 nm, filter cube: 570−620 nm, laser power: 14%, PMT HV: 700, PMT Offset: 0) and DiD (laser wavelength: 640 nm, filter cube: 650−700 nm, laser power: 5%, PMT HV: 420, PMT Offset: 0).

Analysis of endocytosis pathways

To examine endocytic pathways involved in the micellar uptake, the cellular uptake was evaluated in the presence of various inhibitors including 20 µM chlorpromazine, 100 µM dansylcadaverine, 40 µM nystatin, 6 µM filipin III, 2 mM methyl-β-cyclodextrin, 20 µM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), 0.2 µM cytochalasin D and 0.2 µM wortmannin. These concentrations were determined based on the results of cell viability assay (more than 90% viability) and morphological observation. Colon-26 cells were pre-incubated with endocytosis inhibitors for 1 h, then DiD-loaded micelles were added, and the cells were incubated for another 30 min at 42 °C. The fluorescence intensity of the lysate was measured as described above.

In vivo targeting of the DiD-loaded micelles

All animal experiments were carried out in accordance with the Guidelines on Animal Experimentations of Keio University (Approval No. A2021-043). Female 6-week-old BALB/cCrSlc mice were purchased from Sankyo Labo Service (Tokyo, Japan). The mice were housed in the animal care facility with food and water ad libitum and were used for experiments at 7–8 weeks old (17–19 g body weight). DiD-loaded micelles in PBS (5.0 mg/mL) were injected via the tail vein (200 µL/ mouse) using a 1.0 mL insulin syringe with a 29 G needle (Terumo, Tokyo, Japan). At 1 min after the injection, the left ear was heated at 42 °C (setting: the first gear) for 30 min using an electric hand warmer (XY-101; ZeRay). During the procedure, mice were anesthetized with isoflurane and placed face up on a heating pad (Heater Mat type III; Natsume Seisakusho, Tokyo, Japan). The mice were imaged using an in vivo imaging system (IVIS Lumina III; PerkinElmer) for detection of DiD fluorescence (excitation filter: 620/20 nm, emission filter: 670/40 nm, exposure time: 5 s, binning: small, F/Stop: 2, lamp level: high, subject height: 1.5 cm). The fluorescence signal was automatically superimposed on the gray-scale photograph of each mouse using the provided software.

At 24 h after the injection, both ears were collected, washed with PBS, and either snap-frozen in liquid nitrogen for the extraction of DiD or embedded in OCT compound (Sakura Finetek, Tokyo, Japan) and frozen in a dry ice-hexane for section preparation. All tissue samples were stored at −80 °C until further evaluation. The tissue samples were minced with scissors in RIPA buffer (2 mL/g tissue), and homogenized using a BioMasher II (Nippi, Tokyo, Japan). DMSO (18 mL/g tissue) was added to the tissue lysate and the mixture was incubated at 50 °C for 24 h. After centrifugation at 15,000 × g at 20 °C for 5 min, 200 µL of the supernatant was transferred to a 96-well black plate and the fluorescence intensity was measured using a Nivo plate reader for DiD detection (excitation filter: 600/10 nm, emission filter: 640/30 nm, dichroic mirror: D565, measurement time: 2500 ms). Standard samples were prepared for each micelle to estimate the value corresponding to 1% injected dose: the supernatant from untreated mice were mixed with micelle solution.

Section preparation and immunohistochemistry

The embedded samples were sectioned into 10-µm slices using a cryostat (CM1950; Leica Biosystems, Tokyo, Japan) at −20 °C and mounted on slides (CREST coated slide; Matsunami Glass, Osaka, Japan). The frozen sections were washed twice with PBS at 4 °C for 10 minutes, fixed in 4% paraformaldehyde phosphate buffer solution (Nacalai) at 4 °C for 10 min, and blocked with 3% bovine serum albumin in PBS for 1 h at RT. Subsequently, the slides were incubated with anti-mouse CD31 antibody conjugated with Alexa488 (Clone: 390; BioLegend, San Diego, CA) at 1:50 dilution at 4 °C for 16 h. After washing twice with PBS at room temperature for 5 min, sections were stained with DAPI solution at room temperature for 15 min and covered with mounting medium. Fluorescence images were acquired using an FV3000 confocal fluorescence microscope; ×40, DAPI (laser wavelength: 405 nm, filter: 430−470 nm, laser power: 3%, PMT HV: 420, PMT Offset: 0), Alexa488 (laser wavelength: 488 nm, filter: 500−540 nm, laser power: 5%, PMT HV: 550, PMT Offset: 0) and DiD (laser wavelength: 640 nm, filter cube: 650−750 nm, laser power: 5%, PMT HV: 500, PMT Offset: 0).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The data that support the findings of this study are available from the corresponding author, K.H., upon reasonable request.

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Acknowledgements

This work was supported in part by JSPS KAKENHI Grant Numbers JP23H02613, JP21H05262, and JP23K17389 to K.H., a grant from the Japan Agency for Medical Research and Development (AMED) (JP23ak0101182h0003, JP23wm0325046s0103, and JP23gm1510012s0201) to K.H., JST CREST to K.H., Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering, The Uehara Memorial Foundation to K.H., Program for the Advancement of Next Generation Research Projects (Keio University) and Academic Development Fund (Keio University Academic Development Funds) to K.H.

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Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo, Japan
Sota Yamada, Eita Sasaki, Hisashi Ohno, Kenichi Nagase & Kenjiro Hanaoka

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Sota Yamada
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Eita Sasaki
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Hisashi Ohno
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Kenichi Nagase
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Kenjiro Hanaoka
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Contributions

Sota Yamada: Conceptualization, data curation, formal analysis, investigation, methodology, validation, visualization, writing—original draft. Eita Sasaki: Visualization, Writing—review and editing. Hisashi Ohno: Writing—review and editing. Kenichi Nagase: Visualization, Writing—review and editing. Kenjiro Hanaoka: Funding acquisition, project administration, supervision, writing—review and editing.

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Correspondence to Kenjiro Hanaoka.

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Communications Chemistry thanks Kanjiro Miyata and the other anonymous reviewers for their contribution to the peer review of this work. Peer review reports are available.

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Yamada, S., Sasaki, E., Ohno, H. et al. Thermo-responsive targeting of polymeric micelles by controlling the cellular uptake based on the change of their surface arginine density. Commun Chem 8, 325 (2025). https://doi.org/10.1038/s42004-025-01707-8

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Received: 22 April 2025
Accepted: 12 September 2025
Published: 04 November 2025
Version of record: 04 November 2025
DOI: https://doi.org/10.1038/s42004-025-01707-8