NIR-Responsive Polypyrrole-Functionalized Fibrous Localized

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Biological and Medical Applications of Materials and Interfaces

pH/NIR-Responsive Polypyrrole Functionalized Fibrous Localized Drug Delivery Platform for Synergistic Cancer Therapy Arjun Prasad Tiwari, Tae In Hwang, Jung-Mi Oh, Bikendra Maharjan, Sungkun Chun, Beom Su Kim, Mahesh Kumar Kumar Joshi, Chan Hee Park, and Cheol Sang Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17664 • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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ACS Applied Materials & Interfaces

pH/NIR-Responsive Polypyrrole Functionalized Fibrous Localized Drug Delivery Platform for Synergistic Cancer Therapy Arjun Prasad Tiwari1, Tae In Hwang1, Jung-Mi Oh2, Bikendra Maharjan1, Sungkun Chun2, Beom Su Kim3, Mahesh Kumar Joshi1,4, Chan Hee Park1,5*, Cheol Sang Kim1,5* 1

Department of Bionanosystem Engineering, Graduate School, Chonbuk National University,

Jeonju 561-756, Republic of Korea 2

Department of Physiology, Chonbuk National University, Jeonju 561-756, Republic of Korea

3

Carbon Nano Convergence Technology Center for Next Generation Engineers, Chonbuk

National University, Jeonju 561-756, Republic of Korea 4

Department of Chemistry, Tri-Chandra Multiple Campus, Tribhuvan University, Kathmandu,

Nepal 5

Division of Mechanical Design Engineering, Chonbuk National University, Jeonju 561-756,

Republic of Korea *(Cheol Sang Kim) E-mail: [email protected], *(Chan Hee Park) E-mail: [email protected]. Tel.: +82-63-270-4284. fax: +82-63-270-2460.

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Abstract Localized drug delivery systems (LDDS) are a promising approach for cancer treatment, because they decrease systematic toxicity, and enhance the therapeutic effect of the drugs via site-specific delivery of active compounds and possible gradual release. However, the development of LDDS with rationally controlled drug release and intelligent functionality holds great challenge. To this end, we have developed a tailorable fibrous site-specific drug delivery platform functionalized with pH and NIR-responsive polypyrrole (PPy), with the aim of cancer treatment via a combination of photothermal ablation and chemotherapy. Firstly, a paclitaxel (PTX)-loaded polycaprolactone (PCL) (PCL-PTX) mat was prepared using electrospinning, and subsequently in situ membrane surface-functionalized with different concentration of PPy. The obtained PPy functionalized mats exhibited excellent photostability and heating property in response to NIR exposure. PPy coated mats exhibited enhanced PTX release at pH 5.5 environment, when compared to pH at 7.4. Release was further accelerated in response to NIR in both conditions, however, the superior release was found in pH 5.5 compared to pH 7.4, indicating a dual stimuliresponsive (pH and NIR) drug delivery platform. More importantly, the 808 nm NIR irradiation enabled markedly accelerated PTX release from PPy coated PCL-PTX mats, and slowed and sustained release following termination of laser irradiation, confirming representative step-wise drug release properties. PPy coated PCL-PTX mats presented significantly enhanced in vitro and in vivo anticancer efficacy under NIR irradiation, when compared with PPy coated PCL-PTX mat not exposed to NIR or uncoated mat (PCL-PTX). This study has thus developed a promising fibrous site-specific drug delivery platform with NIR and pH-triggering that notably utilizes PPy as a dopant for synergistic photothermal-chemo therapy.


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Keywords: Polypyrrole, Electrospinning, Fibrous membrane, pH/NIR-responsive, Photo-chemo therapy 1. Introduction Recently, the hybridization of effective treatments can offer combinational therapy with higher success rates, increased survivability, and improved quality of life. Several therapeutic particulate drug delivery systems that integrate chemotherapies with photothermal agents, such as gold nanomaterials,1-2 carbon nanomaterials,3-4 gold/copper nanohybrids,5 and conjugated polymers,6-7 show great potential in cancer treatment. NIR-induced photothermal therapy (PTT) is presently being widely investigated and developed as an alternative to conventional cancer treatment modalities, due to its being less invasive in nature, and offering high selectivity.1, 3-4, 8 NIR falls in the 650-1450 nm region of the spectrum with the lowest absorption in tissue, therefore enabling maximum tissue penetration (5-30 mm) and use in PTT applications.9 PTT agents absorb NIR light,and dissipate the absorbed energy through heating, which induces a temperature rise in the local milieu that results in irreversible cell damage.7, 10 Nevertheless, PTT alone is unlikely to eradicate tumor cells, because of the heterogeneous distribution of heat in the tumor. Moreover, there is the possibility of residual surviving cells after therapy spreading to distant organs, resulting in cancer metastasis or tumor remission.11-12 On the other hand, chemotherapy, a common treatment for many cancer types, cannot by itself sufficiently eliminate tumors; rather, it induces many severe health issues, such as drug resistance and toxic side effects, due to non-specific distribution.3-4, 12-13 Photothermal agents alone or in combination with other drugs, are generally encapsulated in polymers or protein materials as nanoparticles,1, 6, 14 liposomes,4 and micelles,12, 15 and subsequently delivered via systematic circulation. Chen et al.6 have used an imageable and photothermal human serum albumin (HSA) indocyanine green (ICG)



paclitaxel (PTX) (HSA-ICG-PTX) nanoparticle to treat subcutaneous and metastatic breast cancers, and the combination therapy achieved excellent synergistic therapeutic efficacy. However, a series of reports have shown the particulate drug delivery system has several drawbacks, such as relative instability, faster renal clearance, overaccumulation in non-targeted tissues, lack of control in drug release and distribution, and the inducement of many inevitable adverse effects.13, 16-17 Moreover, these systems often encounter multiple concerns, such as long fabrication processes, complex compositions, and limiting loading capacities. Therefore, developing a combinational therapeutic platform through facile controllable fabrication for a temporally active drug delivery system is highly desirable. Recently, a series of studies have shown that electrospun mats13, 16, 18-19 and hydrogels20-21 can be used for site-specific drug delivery. Hydrogels offer promise for localized drug delivery, because of their unique physicochemical characteristics, which can mimic those of living tissues.20-21 However, the solidification of the liquid hydrogel in vivo is sometimes inconvenient, an initial burst of drug may occur during the lag time between the injection and the formation of the solid hydrogel, and poor mechanical properties and stability limit its applications.22-23 Drug-loaded electrospun fibrous mats have attracted a great deal of attention as implantable devices for solid cancer chemotherapy or after surgical removal of solid tumors, due to their higher drug encapsulation efficiency, better stability, and enhanced temporal drug discharges compared to other drug formulations.13, 17-19, 24-27 More importantly, the possibility of local drug delivery in cancer therapy using electrospun materials allow oral or systemic drug applications to be avoided, leading to a decrease in the inevitable adverse effects associated with chemotherapeutics. Zhang et al.18 have shown that 5- fluorouracil and oxaliplatin loaded electrospun polylactide fibers are more effective in reducing tumor sizes, compared to free drugs administered systematically.


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Recently, Zhou and co-workers17 have established a well-defined LDDS, by combining activetargeting micelles with implantable polymeric nanofibers. They have found greatly reduced tumor sizes, without any noticeable chemo-associated side effects. However, poor response and therapeutic outcome to chemotherapy due to rapidly acquired drug resistance and distant metastasis can lead to treatment failure.3-4, 12-13, 15 Several other reports have acknowledged that the incorporation of an external triggering system, such as light or pH, into an implantable matrix can achieve higher therapeutic efficacy against tumors, and minimize the toxicity on normal tissues, thus overcoming the shortcomings of conventional LDDS.13,

15-16

External triggering

factors are not only involved in the direct killing of cancer cells, but also sensitize the cells to acquire more chemo,3, 15-16 and thus offer great promise to overcome the current challenges of chemo resistant cancer treatments. Many researchers have reported that using gold nano rods13 and rare earth metals,16 along with chemo drugs in electrospun membranes, to achieve synergistic therapeutic efficacy. They found that their LDDS platform showed enhanced anticancer properties, due to the synergistic effect of NIR-assisted phototherapy and chemotherapy. Nevertheless, the majority of PTT agents are usually inorganic and nonbiodegradable, which result in them staying longer in the body, even after clinical treatment, and can induce potential hazards because of their long-term toxicity. Furthermore, these PTT agents often have many limitations over longer periods of time and are repetitively used because of melting and fragmenting under laser.28-29 In this context, the development of LDDS comprised of biocompatible organic compounds with excellent photothermal behavior along with a potent anticancer drug is desirable. PPy is a biocompatible organic compound that behaves as a photothermal agent with excellent photo stability and photo conversion performance under NIR laser.29-30 In addition, PPy shows



pH responsive behavior: it undergoes swelling in low pH (C=O), encouraging PPy adherence. Meanwhile, the PPy particles undergo homogeneous nucleation and form in arrays in a matrix via mutual attraction due to van der Waals force or chemical bonding.39 With increasing polymerization time, an increased population of nanoparticles forms a network that gradually thickens, resulting in a continuous coating layer throughout the substrates. Moreover, with increased PPy concentration results in a formation of a combination of thin film and particle layers on the substrate as shown in Fig. 2. To demonstrate the PPy coating, we removed the core layer (PCL-PTX), using a sacrificial template method.40 Cross-section images indicate clearly that PPy forms a shell layer of 150-200 nm thickness over the PCL-PTX fibers (Fig. S1). The combined nature of the inherent aggregation of PPy particles in solution, poor solubility in common solvents, and highly conductive nature of the particles limits direct electrospinning.41 To this end, homogeneous coating of electrospun mats by PPy via in situ polymerization can be an alternative way to fabricate mats with embedding or functionalization by PPy particles due to the easy processability, and cost-effectiveness.



Figure 2. FE-SEM images (top and middle) and fiber diameter histograms (bottom) of different fibrous membranes. We have varied the concentration of pyrrole monomer for functionalization, thus obtaining the PPy coated PCL-PTX fibers with different morphologies in terms of fiber diameter and surface topography (Fig. 2). PPy doped mats obtained from 0.02, 0.04, and 0.06 M are referred to as PCL-PTX/PPy1, PCL-PTX/PPy2, and PCL-PTX/PPy3, respectively. The average fiber diameters of PCL-PTX/PPy1, PCL-PTX/PPy2, and PCL-PTX/PPy3 were 520±177, 588±211, and 651±198 nm, respectively. Furthermore, PPy coating in various mats was measured quantitatively, and found 4.0, 5.9, and 7.3 % loading for PCL-PTX/PPy1, PCL-PTX/PPy2, and PCL-PTX/PPy3, respectively (Fig. S2). These results suggest that gradual increasing of pyrrole concentration during polymerization is directly leading to increasing amount of PPy deposition


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in a pyrrole concentration-dependent manner. Therefore, it is not surprising to observe larger diameter in the PCL-PTX/PPy fibers compared to the PCL-PTX fibers. Further characterization of composites membranes was performed using FT-IR, XRD, and DSC. Figure 3 (A) shows the FT-IR spectra. Absorption peaks appearing at 2,945, 1,720, and 1,239 and 1,166 cm−1 for all mats PCL-PTX, PCL-PTX/PPy1, PCL-PTX/PPy2, and PCL-PTX/PPy3 in Fig. 3A are attributed to the -CH2 stretching, CO stretching, and C–O–C stretching of PCL, respectively.38, 42 The additional strong band in composite fibers at 1,540 cm-1 was attributed to the existence of a stretching pyrrole ring (C–C band).43 Similarly, peaks at 784 cm-1 in the composite membrane were assigned to pyrrole ring vibrations.43 Thus, inclusion of these characteristic peaks of PPy in the composite clearly establishes the presence of PPy. FT-IR spectra of pure PTX in Fig. S3 showed characteristic peaks centered at 1,065 cm−1 (aromatic C and H bonds), 1,239 cm−1 (CO stretching), and 1,720 cm−1 (CO stretching).44 Among them, the 1,065 band distinctly appeared in the composite fibers (Fig. 3A). However, other typical bands for PTX were not obvious, mainly because of the overlap with the bands for either the PCL or PPy. Moreover, PPy functionalization of the PCL-PTX fibers leads to a clear reduction in the intensities of all peaks corresponding to the PCL-PTX matrix in a concentration-dependent manner. Additionally, PPy-coated nanofibers showed a characteristic absorption band at 210 nm in UV-VIS spectroscopy (Fig. S4 ), further confirming the presence of PTX in the composite fiber.45



Figure 3. Characterization of the membranes. (A) FT-IR spectra, (B) XRD patterns, (C) DSC thermograms and (D) stress-strain curves. Figure 3 (B) shows the XRD curves for the specimens. PPy has a broad peak at lower diffraction angle 2θ = ∼24°, which indicates its amorphous nature with no clear indication of crystallinity. Similarly, according to our previous works,19, 44 PTX upon loading into the nanofiber did not show any crystalline peaks. However, the composite fibers showed strong peaks at (21.4 and 23.7)° (Fig. 3 B), which were attributed to a crystalline diffraction peak for the PCL chain segments.46-47 Moreover, these crystalline peak intensities gradually decreased when increasing


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PPy particles were deposited, because deposition reduced the crystallinity degree of PCL. DSC study was also performed (Fig. 3C), in order to investigate the effect of PPy on the thermophysical property of the PCL-PTX matrix. The PCL-PTX membrane showed an endothermic peak around 62 °C, which is associated with the melting point of PCL.46 Figure 3C shows that the influence of PPy on the composite can be identified from the slight decrease of melting temperature from 62 ºC for PCL-PTX to 60.7, 59.4, and 55.4 °C for PCL-PTX/PPy1, PCL-PTX/PPy2, and PCL-PTX/PPy3, respectively. This decrease of melting point is related to decreasing crystalline nature of the composite, which is attributable to the covering of PCL-PTX fibers by non-crystalline PPy as observed in FE-SEM imagery (Fig. 2). Table 1. Mechanical properties of the different membranes.

Elongation at break Young's

Modulus Tensile

Strength

Materials (%)

(MPa)

(MPa)

PCL-PTX

480.2 ± 23.1

9.2 ± 1.5

10.3 ± 0.9

PCL-PTX/PPy1

324.2 ± 21.3

6.8 ± 0.8

9.2 ± 0.4

PCL-PTX/PPy2

247.4 ± 18.5

7.6 ± 1.3

8.5 ± 0.5

PCL-PTX/PPy3

187.3 ± 17.4

6.6 ± 1.3

5.9 ± 0.8

Note: Each sample was evaluated for three times. The mean values and corresponding standard deviation is displayed. A successful LDDS platform requires the ability to withstand in vivo stresses of significant magnitude and temporal loading regimes from the time of implantation. Lacking mechanical



integrity at the time of implantation will likely lead to treatment failure, or adverse integration of the construct with surrounding tissue.48 To confirm that the membranes are sufficiently mechanically strong to remain coherent in vivo and safely contain a depot concentration of drug, the mechanical stability of the membranes was evaluated. Figure 3 (D) shows stress-strain curves for all the tested samples. Similarly, Table 1 summarizes all the mechanical properties, including elongation at break, Young’s moduli, and tensile strength. The results exhibited that the deposition of PPy into PCL-PTX fiber decreases the mechanical properties of the PCL-PTX membrane. As shown in Fig 4D, membranes indicated a linear stress decreasing gradually from 10.3 for PCL-PTX, to 9.2, 8.5, and 5.9 for PCL-PTX/PPy1, PCL-PTX/PPy2, and PCLPTX/PPy3 mats, respectively (Fig. 3D and Table 1). Also, we can observe a similar trend in the case of the elongation of break (Fig. 3D and Table 1), indicated by strain. These are typical behaviors for polymers filled with organic or inorganic fillers since fillers reduce the drawability of polymers.49-50 Increased deposition of particles on the fiber surface caused fibers to become less mobile, as a consequence reducing the ability of particles to dissipate energy during stretching,51 which could make the particles-coated fibers show lesser tensile stress and strain (elongation), compared to the uncoated one. The improved tensile stress and strain of PCLPTX/PPy1 fiber compared to the other PPy coated fibers might have been due to the homogenous distribution of PPy particles on the fiber surfaces, when compared to the latter. Studies reported that the size and distribution pattern of embedding particles influence the mechanical properties of the electrospun membrane.46, 52-53 Similarly, PCL-PTX has a slightly higher Young’s modulus than the PPy coated fibers. Table 1 shows a Young’s modulus of 6.8, 7.6, 6.6, and 9.2 MPa for PCL-PTX/PPy1, PCL-PTX/PPy2, PCL-PTX/PPy3, and PCL-PTX mats, respectively. However, these results are in contrast with other reports which have shown


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increases in Young’s moduli attributed to the increasing stiffening effect after coating with filler.49, 53 The decrease in the Young’s modulus of the PCL-PTX when coated with PPy in this study can be attributed to the lower crystallinity of the PCL-PTX/PPy composites due to presence of non-crystalline PPy in the mats. Crystalline properties of a polymer are highly linked to its mechanical strength, and a higher crystallinity will usually give polymer material stronger tensile properties.46 The mechanical properties of materials are in accordance with the DSC results which showed that PCL-PTX/PPy mats have lower crystallinity compared to their pure PCL-PTX counterparts. Moreover, some aggregation of these PPy particles on the fiber surface could play a role in reducing the stiffness, as indicated by the Young’s moduli of the composites. The mechanical properties, as shown by the as-fabricated fibrous membranes in the range of 6– 10 MPa (tensile strength) and appropriate Young’s moduli, revealed that these membranes are high enough to meet the requirements to support soft tissue and organ47 thereby endorse as an implantable therapeutic device. 3.2 Photothermal ability of membranes



Figure 4. Photothermal properties. (A) NIR thermographic image of PPy coated PCL-PTX mat (PCL-PTX/PPy1) under irradiation, (B) Heating curves of different PPy coated PCL-PTX mats under NIR laser power 0.5 W/cm2, (C) Stability assay; elevated temperature profiles of PPy coated PCL-PTX mat (PCL-PTX/PPy1) over ten cycles of exposure (laser on time:5 min; laser off time: 30 min), and (D) FE-SEM image of the PPy coated PCL-PTX mat (PCL-PTX/PPy1) after 10 cycles of laser on/off process. NIR laser (8.0nm) of power density of 0.5 W/cm2 was used for all experiments. Figure 4A presents NIR thermographic image of PPy coated membrane under laser irradiation. Similarly, Figure 4B shows the potential of the photothermal ability of the different fiber meshes. For example, PCL-PTX/PPy1, PCL-PTX/PPy2, and PCL-PTX/PPy3 mats exhibited quick rises


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up to 43–45, 52.4, and 62 °C, respectively, at a laser of 0.5 W/cm2 in 300 s, while the uncoated showed no heating ability (Fig. 4B). Photothermal properties of the given mats under laser irradiation with different power densities are summarized in Table S1. Interestingly, PPy coated mats showed dramatically higher peak temperature elevation in the first 50 seconds, where it reached a threshold and remained nearly constant up to our study period (5 min). This is in contrast to the irradiation of particulate suspension containing photothermal agents where the temperature of the system increases gradually. There are many small heating centers (photothermal agents) uniformly distributed in a solution. When irradiation is employed, the photothermal agents immediately dissipate the heat to adjacent solution thus there may not develop temperature gradient in a solution. But, in this study, heating center (PPy coated mat in a solid state) is confined in a small area of petridish containing PBS solution. Therefore, NIR irradiation of PPy coated mats induced a temperature gradient into the system once the PPy is saturated with vibrational energy.54 This leads to spontaneous heat flows from the hightemperature region (irradiated mat) to the surrounding low-temperature region (medium),54 hence the stable temperature after some time. The different mats have shown various peak temperatures in the same period of time, is associated with the variable PPy contents in the mats. Moreover, after the laser is turned off, fast temperature decreases were observed within a minute (data not shown) indicating that PPy coated mats can dissipate the heat stored rapidly. Quickly triggered hyperthermia in the NIR-on state followed by an immediate temperature drop in the NIR-off state is beneficial for localized treatment, as it can reduce the therapy time and possibly increase therapeutic outcome. Based on the heating property of the different meshes in both pH condition (table S2), PCL-PTX/PPy1 can be a suitable candidate for in vitro photothermal treatment, due to showing the appropriate temperature range (42-45 °C) for conventional



hyperthermia treatment using low laser power. The key prerequisite to trigger cell death from conventional hyperthermia treatment is a (42–45) °C temperature.55 Meanwhile, other PPy coated membranes can be used in ablation therapy, which is conducted at high temperatures (5070 °C),55 due to their high photothermal conversion efficiency. Hereafter, PCL-PTX/PPy1 mat is referred to as PPy coated PCL-PTX mat (PCL-PTX/PPy), which is utilized for further assessments. Furthermore, to consider the utilization of these mats in the acidic extra- cellular microenvironment, we evaluated the photothermal activity of the representative PPy coated mat (PCL-PTX/PPy1) in pH 5.5 condition at different time intervals (0, 6, 48 and 168 h) and further compared these values with the photothermal activity of PCL-PTX/PPy mat in pH 7.4 condition. Data are presented in Table S2. Results demonstrated that activity was not influenced by the pH changes for 0 and 6 h. Nevertheless, slightly decreased photothermal performance was noticed when the test was carried out at 48 and 168 h in pH 5.5 condition compared to the pH 7.4 condition. This result may be associated with the increased swelling of the PPy coated mats at pH 5.5 buffer condition (Fig. S6A). Swelling is due to water absorption which may have reduced the conductivity of the fiber and thereby showing decreased photothermal performance.39 Cancers cells, due to their aggressive behavior, may not be eliminated in a single NIR laser treatment; therefore, proper evaluation of the photothermal stability of membranes under repeated NIR laser irradiation is an equally important task. In this study, PCL-PTX/PPy fibrous mat was irradiated by NIR laser (0.5 W/cm2) for 5 min (laser on), followed by allowing to room temperature without NIR laser irradiation for 30 min (laser off). This cycle was repeated ten times. Figure 4C shows that no changes of elevated temperature were observed throughout the whole cycles i.e. the temperature stays between 43–45 °C. Further, SEM imagery (Fig. 4D) of the irradiated samples after ten cycles of irradiation was taken, to confirm any changes in the


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fiber morphology, orientation, and distribution of PPy particles. Based on the image, the fibers’ surface was found to be still intact (Fig. 4D), and there were no significant changes in fiber morphology and surface topography, when compared to the mat not exposed to NIR (Fig. 2 (FESEM image of PCL-PTX/PPy1 mat)). Similarly, UV-VIS-NIR was employed to study the effect of NIR laser on the absorption spectra of PPy coated mat before and after cycles of treatment. As shown in Fig. S7, both irradiated and non-irradiated PCL-PTX/PPy mats exhibited a broad absorption band in the NIR region (>760 nm), which can be attributed to the polaronic and bipolaronic transitions of PPy.56 The peak position and intensity of the 10-cycle irradiated sample is well consistent to that of the non-irradiated sample, suggesting excellent stability of the PPy coated sample, which is in good agreement with results of other stability assays (Fig. 5C and D). These results suggest that PPy coated membrane has excellent photo stability, which further proves its potential application for the photothermal ablation of tumor cells, allowing its long time and repetitive use. Gold nanomaterials are widely used photothermal agents for the diagnosis and treatment of cancer.1-2,

11, 13

However, it often has many limitations, such as

decreasing thermal stability, melting and fragmenting for longer time, and repetitive use.28-29 In this scenario, PPy could be an appropriate candidate for photothermal treatment in vivo when embedded in implant materials. We also measured the temperature of the solution containing sample (Fig. S5) during irradiation, in which the result showed no apparent increase of the solution temperature, i.e. the temperature stays between (25–28) °C, suggesting that the photothermal effect of PPy may be effectively utilized for killing the cancer cells only, without significant heating the surrounding environment to a high apparent temperature. 3.3 Swelling study



Swelling of the fibrous mats in PBS of different pH were evaluated by measuring the weight of the mats at designated times. Increased weight can be explained by swelling of the matrix due to medium absorption.57 Figure S6A shows PCL-PTX/PPy mats exhibited increased weight in the same ratio in first 2 h in both pH conditions. Coating of PCL-PTX mat by PPy increases the surface roughness thereby facilitating the water absorption.58 Interestingly, the pH of the solution affected the weight of the fibrous mats after 6 h. The enhanced weight of the PPy coated PCLPTX mat that were kept in pH 5.5 was significantly increased after 48 h (*p

NIR-Responsive Polypyrrole-Functionalized Fibrous Localized

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