Introduction

Self-assembly is a natural process in which disordered components are spontaneously assembled into a certain structure or pattern1. Non-covalent interactions such as hydrogen bonding, van der Waals interactions, and electrostatic interactions are major driving forces of self-assembly2,3,4. In particular, nano-sized and self-assembled structures are one of the most useful materials to provide various functionalities in biomedical applications5,6,7,8. For instance, the nano-sized and self-assembled structures are highly useful for the delivery of hydrophobic anti-cancer drugs because of their facile formulation. In addition, these structures exhibit the unique property of accumulating within the tumor site because of their enhanced permeability and retention (EPR) effect9,10,11,12,13. Therefore, various formulations such as liposomes, polymeric micelles, polymersomes, and inorganic-organic hybrids have been developed and evaluated over the course of decades for use in biomedical applications14,15,16,17,18. Doxorubicin (DOX) is a one of the most commonly used anti-cancer chemotherapeutics for various types of cancer, including blood, breast, lung, and ovarian cancers19,20,21,22. The molecular structure of DOX comprises two characteristic parts: the hydrophobic tetracycline ring and an amine-functionalized sugar. Although DOX is administered intravenously as an aqueous solution of the hydrochloride salt, several studies have reported hemolytic activity of DOX, resulting from its cationic charge and hydrophobicity23,24,25. To improve the blood compatibility of DOX and extend its circulation time in the blood, liposomal formulations have been introduced and recently commercialized, including Doxil®, Myocet®, and Caelyx®26,27,28,29. However, there are concerns about non-specific leakage from liposomes and polymeric micelles during blood circulation, which can be driven by large dilution and decreased stability under high salt conditions. Therefore, a more stable nanoparticle system is still needed for the efficient delivery of DOX to enhance the drug's efficacy and limit its side effects30,31,32. In that regard, the distinct chemical features of DOX, such as its hydrophobicity and cationic charge, have been exploited for loading DOX into delivery cargos such as the hydrophobic core of liposomes, polymeric micelles, or nanoparticles18,33,34,35,36,37. Further, DOX has been adsorbed on the surface of carbon nanomaterials and intercalated into specific DNA sequences via π-π interaction17,38,39,40,41. Charge interaction with negatively charged materials such as the surface of silica or polyanions is another common strategy utilized for loading DOX42,43. However, such systems impede the precise control of drug loading, and complicated chemical modification is required in many cases. Here, we approached this issue from a different direction from the approaches used in previous studies. The molecular structure of DOX contains a 9-(2-hydroxyacetyl) group and the 9S-hydroxyl group, which comprise a 1,3-diol conformation. Notably, phenylboronic acid (PBA) has a remarkable binding affinity toward 1,2-cis-diols and 1,3-diols via the formation of a boronic ester44,45,46,47. Therefore, strong binding between PBA and DOX was expected and provided clues to a new strategy for formulatinge self-assembled nanostructures.

Recently, our group has reported the self-assembled architecture between PBA-grafted polyethyleneimine (PEI) and sugar-grafted PEI via the formation of a phenylboronic ester, and has found that the resultant products exhibit feasible gene delivery efficacy48. We have also reported nanocomplexes of entangled poly-paclitaxel (pPTX) and poly-cyclodextrin (pCD) via the self-assembly of a CD::PTX inclusion complex for the enhanced aqueous solubility and delivery of PTX40. Extending our previous studies, here we developed a nanocomplex between polymerized PBA (pPBA) and DOX and evaluated that nanocomplex both in vitro and in vivo. PBA was grafted onto the backbone of poly(maleic anhydride) by a spontaneous ring opening reaction to achieve pPBA. Because neither catalysts nor toxic organic solvents were utilized, the synthetic strategy of pPBA demonstrated atom economy and green chemistry49. Through the simple process of mixing aqueous solutions of pPBA and DOX, self-assembled pPBA-DOX nanocomplexes were generated via the hydrophobic interaction between the PBA-DOX pairs. Furthermore, DOX was easily released at the intracellular pH, owing to the pH sensitivity of the boronic ester, and further destruction was observed (Scheme 1). A remarkable therapeutic effect is expected from our rationally designed pPBA-DOX nanocomplexes both in vitro and in vivo: because 1) the circulation time in vivo can be extended by blocking non-specific interactions with blood components, owing to the negative charge from the residual carboxylic group, 2) the suitable size of the nanocomplexes enables effective tumor accumulation via the EPR effect in vivo, 3) the remaining PBA moieties can be exploited as a tumor-targeting ligand, and 4) DOX can be released in response to the intracellular environment. Consequently, a simple but efficient pPBA-DOX nanocomplex would provide a fascinating strategy for the delivery of conventional drugs not only in anti-cancer therapy but also in broader treatment methods.

Figure 7
figure 7

Schematic illustration for formulation and destruction of pPBA-DOX nanocomplex. Formulation of nanocomplex is driven by charge interaction and the boronic ester formation between phenylboronate of pPBA and 1,3-diol of DOX. At an acidic pH, destruction of nanocomplex is induced by decreased affinity of boronic ester, and finally DOX is released.

PowerPoint slide

Full size image

Materials and methods

Reagents

Poly(methyl vinyl ether-alt-maleic anhydride) (pMA; Mn ∼80 000 g/mol), 3-aminophenylboronic acid (PBA-NH2) monohydrate, and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St Louis, MO, USA). FCR648-NH2 was purchased from BioActs (Korea). Doxorubicin (DOX) was purchased from Wako Chemical (Japan). All reagents were used as received without further purification.

Instrumental methods

Transmission electron microscopy (TEM) images were taken using a transmission electron microscope (JEM-2210, JEOL) and analyzed using Gatan DigitalMicrograph software. UV-vis absorbance for the MTT assay was measured using a microplate spectrofluorometer (VICTOR3 V multilabel counter), and fluorescence spectra were obtained from a spectrofluorophotometer (RF-5301 PC, Shimadzu) or multi-well plate reader (SpectraMax® i3, Molecular Devices). Hydrodynamic size and zeta potential were measured using a Zetasizer (Nano S90, Nano Z, respectively; Malvern) at 0.1 mmol/L in Dulbecco's phosphate-buffered saline (DPBS, pH 7.4) or 0.1 mol/L acetate buffer (pH 5.5). The confocal laser scanning microscope (CLSM) images were obtained using an Olympus FV-1000 and analyzed using the Olympus FluoView ver 1.7 software.

Preparation of poly(phenylboronic acid-co-maleic anhydride) (pPBA)

PBA-conjugated pMA (pPBA) was prepared by spontaneous amide coupling of the acid anhydride moiety onto the pMA backbone and -NH2 of PBA-NH2 via a ring opening reaction (Scheme S2). Briefly, 500 mg of pMA (3.2 mmol anhydride eq) and 160 mg of PBA-NH2 (1 mmol) were dissolved in 15 mL of dry dimethyl sulfoxide (DMSO) and stirred at room temperature (RT) overnight. Unreacted succinic anhydride moieties were hydrolyzed by adding 10 mL of 0.1 mol/L NaOH and dialyzed against deionized water for two days (molecular weight cutoff=10 000), followed by lyophilization. The molar ratio of conjugated PBA was calculated by 1H NMR (yield: 91%). 1H NMR (D2O, 300 MHz): 7.7–7.0 (m, Ph, 4xH); 3.8–3.5 (m, −CH−, 1H); 3.5-3.1 (m, −OCH3, 3H); 3.1–2.4 (m, anhydride, 2H); 2.4–1.4 (m, −CH2−, 2H).

For various imaging experiments in vitro and in vivo, red fluorescence dye was conjugated onto the pPBA backbone in the same manner. In brief, 200 mg of pMA, 70 mg of PBA-NH2, and 5 mg of FCR648-NH2 were fully dissolved in 15 mL of dry DMSO and stirred at RT overnight. Then, 10 mL of 0.1 mol/L NaOH was added and dialyzed against water for two days (molecular weight cutoff=10 000), and this was followed by lyophilization. The conjugation ratio of PBA was calculated from the 1H NMR (yield: 95%).

As a control polymer that contains no PBA moiety, pMA was hydrolyzed by NaOH and dissolved in DPBS to obtain hydrolyzed poly(maleic anhydride) (pMAh) solution. For the controlled experiment, [COO−] from the hydrolyzed succinic anhydride subunit was equalized from both pPBA and pMAh solutions.

Formulation of pPBA-DOX nanocomplexes

The pPBA-DOX nanocomplexes were simply prepared by mixing the pPBA and DOX solutions. For nanocomplexes with [PBA]:[DOX] molar ratios of 1:1, 2:1, or 4:1 (nanocomplexes 1, 2, and 4, respectively) containing 2 mmol/L DOX equivalent, 200 μL of 10 mmol/L DOX was added into 100, 200, or 400 μL of 20 mmol/L pPBA, respectively. Then, 100 μL of 10× PBS (pH 7.4) was added to the solution, and the vial was filled to 1 mL with deionized water. For the quenching assay, pPBA was fixed at 1 mmol/L, and the amount of DOX varied. For the quenching assay of DOX, DOX was fixed at 1 mmol/L, and the amount of pPBA varied. For the formulation of dye-labeled nanocomplexes, non-labeled pPBA and FCR648-labeled pPBA were mixed at the ratio of 1:1 and 1:3 for the formulation of nanocomplexes 2 and 4, respectively, to normalize the fluorescence intensity.

Stimuli-responsive drug release

Drug release from nanocomplexes 1, 2, and 4 at pH 7.4 and 5.5 was monitored by the inherent fluorescence of DOX (Ex=495 nm, Em=590 nm). Within a desired time interval, the solution was centrifuged (13 500 r/min, 5 min), and the fluorescence of the supernatant was measured. The amount of released DOX was calculated as [(released drug)/(loaded drug)×100].

In vitro cell viability test

To evaluate the therapeutic efficiency of the nanocomplexes, the cytotoxicity of the nanocomplexes or control samples was measured with MTT assays. Cells (human breast cancer cells, MCF-7 and human prostate cancer cells, PC-3) were seeded on a 96-well culture plate at a density of 8000 cells/well and incubated overnight. Fresh medium was treated with either pPBA at a final concentration of 0 to 40 μmol/L, free DOX, or nanocomplexes 1, 2, and 4 at a final equivalent DOX concentration of 0 to 10 μmol/L. The medium was then incubated with the cells for another 48 h. After incubation, the cell viability was evaluated with MTT assays.

For the MTT assays, cells were washed with DPBS, and the medium was replaced with 200 μL of MTT solution in the medium (0.5 mg/mL). After incubation in the dark for 4 h, the medium was removed thoroughly, and purple crystals were completely dissolved by 200 μL of DMSO. Each sample (100 μL) was transferred into a new 96-well plate, and UV-vis absorbance was measured at 570 nm using a microplate spectrofluorometer (VICTOR3 V multilabel counter). The relative percentages of non-treated cells were used to represent 100% cell viability.

Competition assay in vitro

The PBA-mediated uptake of nanocomplexes was evaluated with competitive assays. Cells (MCF-7 or PC-3) were seeded on a 96-well culture plate at a density of 8000 cells/well and incubated overnight. After being washed with DPBS, the cells were incubated with pre-treated serum-free medium with or without 5 mmol/L PBA-NH2 for 30 min. Then, cells were washed and incubated with serum-free medium treated with 0 to 40 μmol/L pPBA, free DOX, or nanocomplexes 1, 2, and 4 at a final equivalent DOX concentration of 0 to 10 μmol/L for 4 h. Cells were washed and further incubated for 44 h, then the cell viability was measured with MTT assays.

Intracellular uptake of nanocomplexes by confocal laser scanning microscopy (CLSM)

The intracellular uptake of nanocomplexes with or without competitor was analyzed by confocal microscopy imaging. Briefly, MCF-7 or PC-3 cells (100 000 cells/well) were seeded on glass coverslips placed in a 12-well culture plate. Cells were incubated overnight and incubated for 30 min with pre-treated serum-free medium with or without 5 mmol/L PBA-NH2 as a competitor. Then, the medium was replaced with fresh serum-free medium containing 2 μmol/L DOX equivalent of pPBA or nanocomplexes 1, 2, and 4 and further incubated for 4 h. To observe the localization of pPBA, FCR648-labeled pPBA was incorporated and diluted with pPBA to control the fluorescence intensity. Cells were washed with DPBS and fixed immediately with 10% neutrally buffered formalin at 4 °C overnight. Cells on the coverslips were mounted with Vectashield anti-fade mounting medium containing 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) (Vector Labs). The intracellular fluorescence of pPBA-FCR648 and DOX was observed by CLSM at excitation/emission wavelengths of 633/647 and 488/530 nm, respectively.

Hemolysis assay

Hemolysis of each sample was evaluated by measuring the released hemoglobin from the erythrocytes. Briefly, fresh mouse blood was prepared in a heparin tube and diluted tenfold by DPBS, and this was followed by centrifugation (2000 r/min, 15 min) to isolate the red blood cells (RBCs). RBCs were diluted, and samples (pPBA, DOX, nanocomplexes 1, 2, and 4) were added with a final concentration of 1 mmol/L DOX equivalent. The suspensions were incubated at 37 °C for 4 h and then centrifuged (2000 r/min, 15 min) to separate the RBC pellet. The absorbance of the supernatant at 400 nm was measured to minimize the absorbance of released DOX and maximize the absorbance of hemoglobin. Saline and 1× lysis buffer were considered to represent 0 and 100% hemolysis, respectively.

In vivo biodistribution of pPBA-DOX

Nanocomplexes

All animal experiments were approved by the POSTECH Biotech Center Ethics Committee. CT-26 cells (1 000 000 cells/mouse) were inoculated subcutaneously (sc) into the flanks of female Balb/c mice. After the average tumor volume reached approximately 300 mm3, saline, FCR648-labeled pPBA, nanocomplexes 1, 2, and 4 (3 mg/kg DOX) were systemically injected. Nanocomplexes 2 and 4 were formulated with the same molar ratio of FCR648-labeled pPBA and the rest with non-labeled pPBA to normalize the fluorescence signal. At 24 h post-injection, the fluorescence intensities of the mice and tumors were measured with an IVIS spectrum small-animal in vivo imaging system at Pohang Technopark Biotech Center (Califer Lifescience, Hopkinton, MA, USA). For the ex vivo imaging of DOX, saline, free DOX, and nanocomplex 4 (3 mg/kg) were injected, and the epifluorescence of DOX from the tumor was compared using IVIS imaging in the same manner.

Anti-tumor study in a mouse model

CT26/FLuc cells (1 000 000 cells/mouse) were inoculated sc into the flanks of female Balb/c mice. When the average tumor volume reached approximately 300 mm3, the mice were divided randomly into 6 groups (7 mice per group). The mice were then injected with 200 μL of each sample: 1) saline, 2) pPBA, 3) free DOX, 4) nanocomplex 1, 5) nanocomplex 2, and 6) nanocomplex 4 (3 mg/kg DOX at d 0 and 4), and the tumor volume was monitored every day after the systematic injection of the samples. Tumor volumes were recorded following the equation for a prolate ellipsoid of ab2/2, where a is the longest and b is the shortest dimension. Tumor growth was monitored until the tumor was ulcerated, at which time the mice were sacrificed according to the rules of the POSTECH Biotech Center Ethics Committee. All data were represented as the mean±SEM, and all significant differences were analyzed with OriginPro 8. The tumor inhibition rate was calculated as the ratio between the final and initial volumes of the tumor. Perfect tumor inhibition (100%) was set as total growth regression, and 0% tumor inhibition was set as the growth rate of the saline-treated group. For imaging the tumor, luciferin substrate was injected intraperitoneally immediately before in vivo imaging, and the luminescence of the tumor was imaged after 10 min. The image was obtained and analyzed with IVIS.

Results

Preparation and characterization of pPBA

Briefly, polymerized PBA (pPBA) was synthesized by chemical conjugation between 3-aminophenylboronic acid (PBA-NH2) and poly(maleic anhydride) (pMA). The conjugation ratio was 156 PBA moieties per 513 maleic anhydride subunits, as calculated by 1H NMR from the ratio between the aromatic region of PBA and −CH2− of the polymer backbone (Figure S1). The Fourier transform infrared spectra of pMA and pPBA also indicated the successful conjugation of PBA and the hydrolysis of the anhydride, which were represented by the disappearance of the acid anhydride peaks (1830–1800 cm−1 and 1775–1740 cm−1) and the appearance of both carboxylic acid (1730–1700 cm−1) and amide (1680–1630 cm−1) of pPBA (Figure S2)50.

Preparation and characterization of the pPBA-DOX nanocomplexes

For the next step, pPBA-DOX nanocomplexes were readily prepared by simple mixing, on the basis of the strong interaction between the 1,3-diol of DOX and the PBA moiety on pPBA. Three different nanocomplexes (1, 2, and 4) were designed on the basis of molar ratios of 1:1, 2:1, and 4:1 (PBA to pPBA:DOX), respectively, to investigate the function of the residual PBA moiety as a targeting ligand. The sizes of the nanocomplexes were measured at the sub-hundred nanometer scale (Figure 1A). As a control, hydrolyzed pMA (pMAh) was mixed with DOX, thus resulting in severe aggregation and precipitation and indicating that the PBA-diol interaction is a major driving force in the formation of the nanocomplexes (Figure S3). To further validate the formation of boronic ester, the fluorescence of pPBA was measured, because the steady-state quenching of the inherent fluorescence of PBA is induced by the formation of the boronic ester48. The boronic ester was quantitatively formed by mixing pPBA with DOX, thus suggesting that residual PBA moieties exist and may have functioned as a targeting ligand (Figure S4A). In addition, the formation of the hydrophobic core was demonstrated by the fluorescence of DOX because DOX fluorescence is quenched by π–π stacking34. As expected, noticeable quenching of the DOX fluorescence was observed, thereby suggesting π-π stacking of DOX at the hydrophobic core of the nanocomplexes (Figure S4B).

Figure 1
figure 1

Physicochemical characterization of nanocomplexes. (A) Size distribution of nanocomplex 1, 2 and 4. (B) Drug release profile of nanocomplex 1, 2 and 4 at pH 7.4 and 5.5. TEM images of nanocomplex 4 at (C) pH 7.4 and (D) pH 5.5.

PowerPoint slide

Full size image

pH-responsive properties of nanocomplexes

Because the phenylboronic ester linkage shows pH-sensitive behavior, pH-responsive drug release and structural deformation of the nanocomplexes were expected (Scheme S3). At a neutral pH, all three nanocomplexes exhibited a hydrodynamic size of approximately 80 nm, whereas the size distribution increased to approximately 200 nm at an acidic pH (Figure 1A). In addition, TEM imaging confirmed that the condensed structure of nanocomplexes 1, 2, and 4 at neutral pH was deformed at acidic pH, corresponding to the data obtained from dynamic light scattering (Figure 1C, 1D, S3C-D).

Given the change in size distribution, the pH-responsive drug release profile of pPBA-DOX nanocomplexes was measured at pH 7.4 and 5.5 (Figure 1B). At endosomal pH, all three nanocomplexes showed 70%–80% drug release in 24 h. In contrast, 20%, 40%, and 30%, respectively, were released from nanocomplexes 1, 2, and 4 at physiological pH. Interestingly, nanocomplexes 2 and 4 were less pH-responsive, in contrast to the dramatic pH-responsive release of nanocomplex 1, probably as a result of a combinatorial effect of increased charge interaction between the carboxylic group on the pMA backbone and DOX as well as the increased hydrophobicity of pMA at acidic pH (Figure S5B).

In vitro cytotoxicity and tumor targeting

The cytotoxicity of the materials was evaluated in vitro to demonstrate the therapeutic effect of the nanocomplexes (Figure 2). The toxicity of pPBA was negligible because of the highly negative charge of the polymer backbone and biocompatibility (Figure S5B). In the MCF-7 human breast cancer cell line, the IC50 value of free DOX was measured as 3.1 μmol/L, and the IC50 values of nanocomplexes 1 and 2 were 2.3 and 2.1 μmol/L, respectively (Figure 2A). Surprisingly, nanocomplex 4 exhibited an IC50 value of 0.85 μmol/L DOX equivalent, which was 3.6-fold more effective than that of free DOX and was possibly due to the targeting effect of the residual PBA moieties. Similar results were observed in the PC-3 human prostate cancer cell line (Figure 2B).

Figure 2
figure 2

In vitro cytotoxicity of pPBA, DOX, nanocomplex 1, 2 and 4 studied in (A) MCF-7 and (B) PC-3 cell line. *P<0.05, **P<0.01.

PowerPoint slide

Full size image

To verify the targeting effect of the residual PBA from nanocomplexes 2 and 4, ligand competition assays were conducted. As expected, the toxicity of pPBA and DOX was not affected by pre-treatment with PBA (Figure S6). In addition, the cytotoxicity of nanocomplex 1 was not influenced by additional ligands, whereas the cytotoxicity of nanocomplexes 2 and 4 decreased significantly with the pre-treatment of the free ligands (Figure 3).

Figure 3
figure 3

Competition assay in vitro. Cytotoxicity of nanocomplex 1, 2 and 4 studied in (A) MCF-7 and (B) PC-3 cell line with or without pre-treatment of PBA-NH2. **P<0.01.

PowerPoint slide

Full size image

To further investigate the PBA-mediated uptake, confocal microscopy images were taken with or without pre-treatment of free PBA. For quantitative microscopic imaging, some portions of pPBA were replaced by FCR648-labeled pPBA (FCR648-pPBA), and the fluorescence per nanocomplex was equalized with the addition of non-labeled pPBA. As expected, the cellular uptake of nanocomplex 4 was mostly inhibited by the pre-treatment with free PBA, corresponding to the cytotoxic effect observed in the competition assay (Figures 4, S7, and S8). In addition, the uptake of pPBA was not significantly affected by competitive inhibitors, thus indicating that the formation of a condensed structure is an effective factor for efficient uptake (Figure S9).

Figure 4
figure 4

Competition assay in vitro. (A) Nanocomplex 1, (B) nanocomplex 2 and (C) nanocomplex 4-treated MCF-7 cell line with or without pre-treatment of PBA-NH2 were analyzed by confocal microscopy. Blue images represent nucleus, red images represent pPBA in nanocomplexes, and green images represent DOX.

PowerPoint slide

Full size image

In vivo tumor targeting

Before evaluation of the anti-tumor effect of the nanocomplexes in vivo, the blood compatibility of the nanocomplexes was evaluated by hemolysis assays (Figure S10). Significant hemolysis was induced by free DOX, whereas none of the pPBA materials exhibited hemolysis, owing to their negative charge.

Given that the in vitro targeting ability of pPBA materials was confirmed, the in vivo biodistribution was next determined to demonstrate the targeting effect of the nanocomplexes. FCR648-pPBA (4 mmol/L) or FCR648-labeled nanocomplexes of 1 mmol/L DOX equivalence were systemically injected, and their distribution was observed after 24 h by whole-body fluorescence imaging (Figure 5). The accumulation of nanocomplexes was significantly enhanced as the amount of residual pPBA was increased. Quantitatively, nanocomplex 4 exhibited a 2.5-fold stronger signal from the tumor region than did nanocomplex 1. In addition, most of the fluorescence signal from the pPBA-injected mice was observed at the kidney. Similarly, ex vivo images of each tumor showed significant contrast in the epifluorescence (Figure 5B and 5D). Furthermore, an accumulation of DOX was detected by ex vivo imaging of the tumor site, and a higher level of DOX was observed in the tumor treated with nanocomplex 4 h than in the tumor treated with free DOX (Figure S11).

Figure 5
figure 5

(A) In vivo bio-distribution of saline, pPBA, nanocomplex 1, 2 and 4 after 24 h injection and (B) Ex vivo images of corresponding tumors. The average epi-fluorescence from (C) in vivo nanocomplexes treated tumor site and (D) ex vivo corresponding tumors. **P<0.01 vs Nanocomplex 1.

PowerPoint slide

Full size image

In vivo anti-tumor effect

To evaluate the therapeutic effect of pPBA-DOX nanocomplexes in vivo, tumor growth in CT26/FLuc-inoculated allograft mice was monitored after the injection of saline, pPBA, free DOX, and nanocomplexes 1, 2, and 4 (3 mg/kg DOX equivalent) at days 0 and 4 (Figure 6A). The tendencies for tumor growth between saline- and pPBA-injected mice were similar, and certain levels of anti-tumor effect were observed in mice treated with DOX and nanocomplex 1. As expected, nanocomplexes 2 and 4 exhibited the most significant anti-tumor effect, as compared with the other samples. In addition, none of the treated groups showed abrupt changes in body weight (Figure S12A).

Figure 6
figure 6

In vivo anti-tumor study. (A) Tumor growth of CT26/FLuc bearing Balb/c mice. Error bars represent±SEM. n=7. **P<0.01. (B) Representative luminescence image from CT26/FLuc tumor of nanocomplex 1, 2 and 4 injected mice and (C) corresponding ex vivo image.

PowerPoint slide

Full size image

After monitoring of the tumor growth, the viability of the grown tumor was evaluated by assessing the inherent luciferase expression of CT26/FLuc (Figure 6B and 6C). The luciferase activity in the CT26/FLuc tumor was significantly diminished in mice treated with nanocomplexes 1 to 4. In agreement with previous in vitro and in vivo results, the luciferase activities in the tumors from saline- and pPBA-treated mice were similar, whereas the luciferase activity in the DOX-treated group declined significantly (Figure S12C).

Discussion

Drug delivery systems using nano-sized complexes have been developed to improve the aqueous stability of drug molecules and to enhance their accumulation at the tumor site via the EPR effect. For higher efficacy and clinical translation, the nanocomplexes need to be conveniently formulated, to be stable during blood circulation and to be able to internalize target sites, specifically by using targeting ligands. In most cases, however, complicated chemical modifications are required to achieve the criteria mentioned above, thus limiting the reproducibility of results for clinical translation. In this study, the binding of PBA with DOX through their chemical structures provides a simple strategy not only for the formulation of nanocomplexes but also for the targeted delivery system.

From that perspective, we formulated tumor-targeting self-assembled nanocomplexes, using polymerized PBA and DOX. Briefly, polymerized PBA (pPBA) was synthesized by simple mixing of 3-aminophenylboronic acid (PBA-NH2) and poly(maleic anhydride) (pMA) via the spontaneous ring opening reaction of the succinic anhydride moiety, and this was followed by hydrolysis of the remaining anhydride moieties to obtain carboxylic acids. In the next step, pPBA-DOX nanocomplexes were formulated through simple mixing by exploiting the interaction between the 1,3-diol of DOX and the PBA moiety on pPBA. Both passive targeting by proper size (50–200 nm) measured by dynamic light scattering and active targeting by the PBA moiety, which has high binding affinity to sialylated epitopes on tumor cells, were expected51,52,53. To verify the formation of nanocomplexes from the formation of a phenylboronic ester, hydrolyzed pMA (pMAh) was mixed with DOX in the same manner as in the control group. A clear nanocomplex solution was achieved from the pPBA group, whereas severe aggregation and precipitation were observed in all nanocomplexes formulated by pMAh, thus indicating that the PBA-diol interaction is a major driving force in the formation of the nanocomplexes.

As previously mentioned, the formation of a phenylboronic ester bond is highly responsive to environmental pH. The ester bond is particularly labile at acidic pH, such as that in the endosomal environment, whereas the bond itself is stable under physiological conditions. Therefore, a pH-responsive drug release and structural deformation of nanocomplexes were expected. At neutral pH, all three nanocomplexes exhibited a hydrodynamic size of approximately 80 nm, which is suitable for drug delivery. Furthermore, dramatic changes in size distribution were observed in all three nanocomplexes at acidic pH from sub-hundred nanometers to approximately 200 nm, owing to three possible explanations: 1) at acidic pH, boronic ester becomes labile, and DOX is easily released from the hydrophobic core of the nanocomplex; 2) the solubility of DOX is significantly increased at acidic pH because of the protonation of the amine moiety; and 3) the hydrophobicity of pPBA is increased by the protonation of carboxylic acid on the hydrolyzed pMA backbone, as inferred from the decreased size in acidic condition in Figure S5A.

pPBA-DOX nanocomplexes were designed for the effective delivery of DOX by passive targeting from the EPR effect and active targeting from residual PBA moieties. The cytotoxicity of the nanocomplexes was significantly higher than that of the DOX in vitro, possibly as a result of the suitable size for uptake and targeting. To verify the targeting effect by the residual PBA of nanocomplexes 2 and 4, a ligand competitive assay was designed as follows. Free PBA was pre-treated to occupy the sialylated epitope to inhibit the PBA-mediated uptake of the nanocomplexes. No significant differences were observed from the cytotoxicity of nanocomplex 1, regardless of the existence of pre-treatment, thus suggesting that no additional PBA moieties could be utilized as targeting ligands after the formulation of the nanocomplexes. Moreover, a significant inhibition of cytotoxicity was observed from nanocomplexes 2 and 4 pre-treated with ligands. Similar results were obtained by confocal microscopy, thus demonstrating the function of ligand-mediated uptake via the PBA moiety and the sialylated epitope in cancer cells. Additionally, the importance of nanosized structures in cellular uptake was verified by confocal microscopy.

In this study, pPBA-DOX nanocomplexes with a high molar ratio of pPBA were designed to enhance the targeting effect by residual PBA moieties. Moreover, the formation of specific structures has been revealed to be an important factor for successful cellular uptake. Therefore, the efficient delivery of nanocomplexes in vivo was expected. To ensure blood compatibility after introduction in vivo, hemolysis of the nanocomplexes was evaluated. Because of the positive charge and strong hydrophobicity of DOX, a certain level of hemolysis was induced by free DOX. However, none of the pPBA materials exhibited hemolysis because of their negative charge.

For the next step, the biodistribution of the nanocomplexes was monitored by fluorescence imaging to confirm the in vivo targeting effect. The accumulation of nanocomplexes was significantly enhanced as the amount of residual pPBA increased. Quantification by average epifluorescence of the tumor site showed that nanocomplex 4 exhibited a 2.5-fold stronger signal in comparison with nanocomplex 1. Although an equal amount of pPBA (4 mmol/L) was injected, interestingly, relatively low tumor accumulation was observed in the pPBA-injected mice compared with those injected with nanocomplex 4. Instead, most of the fluorescence signal from pPBA-injected mice was observed at the kidney rather than at the tumor site. Similarly, ex vivo images of each tumor showed a significant contrast in the epifluorescence due to the different targeting abilities of the three nanocomplexes. These results indicate that 1) free pPBA is secreted, 2) accumulation at the tumor site requires the formation of certain structures, and 3) accumulation at the tumor site can be enhanced by an additional targeting ligand.

As mentioned above, the formation of nanocomplexes enabled the successful accumulation at the tumor site in vivo through both passive targeting from the EPR effect and active targeting from the residual PBA moieties. Moreover, the nanocomplexes exhibited an excellent anti-cancer effect in vitro. Overall, the results from the in vitro study and the in vivo biodistribution strongly suggest the potential of the nanocomplexes as an efficient anti-tumor agent. From our anti-tumor study in vivo, the tendency of tumor growth between saline- and pPBA-injected mice was similar, thereby indicating that pPBA itself had no therapeutic effect. Because DOX itself is a practical anti-cancer drug, a certain level of anti-tumor activity was observed. However, the systemic administration of DOX still carries the risk of systemic toxicity, owing to the hemolytic property, as demonstrated by the hemolysis assays, and the possibility of non-specific delivery. The tumor growth observed in mice treated with nanocomplex 1 was similar to that in mice treated with free DOX. As expected, nanocomplexes 2 and 4 exhibited the most significant anti-tumor effect compared with the other samples. These results can be explained by the advantages of precisely designed pPBA-DOX nanocomplexes described above: 1) extended blood circulation time due to strong negative charge, 2) successful accumulation at the tumor site from both the EPR effect and targeting effect, 3) enhanced uptake, owing to the existence of residual PBA moieties, and 4) successful intracellular pH-responsive DOX release after uptake. Similar results were obtained by the luciferase assay of grown tumors, thus suggesting the anti-tumor effect of the nanocomplexes at the histological level. In addition, the changes in the body weights of all sample-treated mice indicated that none of the samples induced severe systemic toxicity at the administered doses.

In conclusion, whereas most of the previously designed DOX delivery vehicles have been focused on charge interaction or hydrophobic interaction, our approach takes advantage of a different phenomenon, the specific formation of a boronic ester between the 1,3-diol of DOX and the phenylboronic acid of pPBA. Despite its ease of preparation, our pPBA-DOX nanocomplex exhibited strong anti-tumor effect both in vitro and in vivo. The improved therapeutic effect of the nanocomplexes can be explained by tumor targeting via the EPR effect, active targeting via the enhanced cellular uptake by residual PBA moieties, and cargo release in the intracellular environment. Together, the overall results from the in vitro and in vivo studies suggest promising therapeutic potential of pPBA-DOX nanocomplexes in biomedical applications.

Author contribution

Junseok LEE performed most of the experiments and wrote the draft of the manuscript; Jinhwan KIM designed the overall experiment and wrote the draft of the manuscript; Junseok LEE, Jinhwan KIM, Yeong Mi LEE, and Sooseok IM participated in the animal experiments; Dongsik PARK contributed to the experimental part of the physicochemical characterization; Eun Ho SONG contributed to the chemical synthesis; Hansoo PARK contributed to the experimental design, and provided invaluable guidance; Won Jong KIM supervised the overall project and wrote the manuscript, to which all other authors contributed.