NIR- and thermo-responsive semi-interpenetrated polypyrrole nanogels for imaging guided combinational photothermal and chemotherapy

Abstract

Versatile, multifunctional nanomaterials for theranostic approaches in cancer treatment are highly on demand in order to increase therapeutic outcomes. Here, we developed thermo-responsive nanogels equipped with the efficient near-infrared (NIR) transducing polymer polypyrrole (PPY) for combinational photothermal and chemotherapeutic therapy along with photoacoustic imaging ability. Long-term stability and water-dispersibility of PPY was achieved using semi-interpenetration method for in situ polymerization of PPY into hydrophilic thermo-responsive nanogels. The semi-interpenetrated nanogels of spherical shape and with hydrodynamic sizes of around 200 nm retained the temperature response behaviour and exhibit excellent photothermal transducing abilities in the NIR region. The PPY nanogels served as photoacoustic contrast agents, which allowed determination of biodistribution profiles ex vivo. In addition, we developed a new method for biodistribution determination based on the photothermal response of the nanogels with an accuracy down to 12.5 μg/mL. We examined the ability of the nanogels as photothermal agents and drug delivery systems in vitro and in vivo. We showed that they efficiently inhibit tumor growth with combinational effects of chemotherapeutics and photothermal treatment. Our work encourages further exploration of nanogels as functional stabilizing matrix for photothermal transducers and their application as drug delivery devices in combination with photothermal therapy and imaging.

Introduction

Photothermal transducing agents able to transform light into heat gained increased interest for the application in photothermal therapy (PTT) of cancer [[1], [2], [3]]. In particular, particles able to transform near-infrared (NIR) light between 700-950 nm attracted increased attention since biological tissue shows particularly low absorbance and scattering of light within this range. As a result, the penetration depth of NIR light into tissue is significantly greater than light of other wavelengths, e.g. light of the UV–visible range, and can reach up to several centimetres deep. Taking advantage of this deep penetration, materials able to transduce NIR light to heat offer minimally invasive approaches for cancer treatment. With the use of external laser irradiation they deliver high thermal energy to the cancerous tissue, allow adjustable energy dosing, and precise local control rendering them into efficient agents for thermal ablation of tumors and yet minimizing harm to surrounding healthy tissue [[1], [2], [3]].

Besides inorganic nanoparticles [[4], [5], [6], [7]] and carbon based NIR transducers [[8], [9], [10]], semi-conducting polymers like polypyrrole (PPY) [[11], [12], [13]], polyaniline (PANI) [[14], [15], [16], [17]], and poly(3,4-ethylenedioxythiophene) polystyrene sulphate (PEDOT:PSS) [18,19] emerged as excellent materials for PTT of cancer with high photostability and good biocompatibilities. In particular, PPY based materials were demonstrated to be suitable materials for biomedical applications like biosensors [20,21], biomaterials in tissue engineering [22], and neural prosthetics [23,24] and thus gained increased interest for PTT [25,26]. First reports of nano-sized PPY particles in PTT of cancer indicate promising results in vitro and in vivo by thermal ablation of tumor cells upon exposure to NIR light [11,12,27,28].

The photothermal effect of PPY based materials additionally allow their use as a contrast agent in photoacoustic (PA) imaging enabling particle localization. PA imaging is an emerging non-invasive technique that provides 3D images of absorption-based contrast with high spatial resolution and allows a comparative and quantitative image intensity analysis [1]. PA imaging typically uses short (nanoseconds) optical excitation pulses to induce local thermal expansion of the tissue which translates into acoustic waves. The spatial distribution of the optical absorption, and hence the local abundance of chromophores, is encoded onto the time-course of the waves, which are detected outside the organism or sample. From these data sets, three-dimensional images are reconstructed that show the spatial distribution of the absorbing chromophores within the illuminated volume. As a result, light-to-heat transducing materials increase the PA signal intensity translating these materials into intrinsic theranostic devices enabling imaging guided therapy approaches. For pure PPY nanoparticles the suitability as PA contrast agent was demonstrated by Dai and coworkers who showed the ability to visualize the particles down to a depth of 4 cm in muscle tissue upon exposure to laser light of 808 nm [29]. In the same group, PPY particles were successfully equipped with a gadolinium chelate which was used to confirm the good correlation between PA and magnetic resonance imaging [30].

Intrinsic imaging modalities additionally are advantageous for the determination of biodistribution profiles of new materials, which is a key parameter for the evaluation of new drug delivery agents. With a label-free detection of the particles, one can disclaim additional synthetic modifications which could alter occurring biological interactions after administration.

As PPY in its pure form is water insoluble, particle stabilization is crucial for a use in biomedical applications. In first reports for PPY nanoparticles for PTT applications, stabilization was achieved by wrapping the final particles using the polymers polyvinyl pyrrolidone (PVP) or polyvinyl alcohol (PVA) added during the polymerization process [11,12]. With promising results of initial work in combinational treatments using PTT along with other therapies like chemotherapy and radiotherapy [[31], [32], [33], [34]], it stands to reason that a rational design of the stabilizing matrix e.g. equipping them with stimuli responsive materials can implement new functionalities for the use of the particles in combinational therapy approaches [3].

One possible option is the incorporation of PPY into hydrophilic nanogels, which are three-dimensional cross-linked polymeric particles that emerged as a versatile tool for the encapsulation of guest molecules [35,36]. In particular upon usage of ‘smart’ polymers for their synthesis, nanogels which change their physico-chemical properties in response to internal or external stimuli can serve as effective drug delivery systems with controlled cargo release upon application of a trigger [35,[37], [38], [39], [40]]. For example, we recently developed thermo-responsive nanogels based on the temperature-responsive polymers poly-(N-isopropyl acrylamide) (PNIPAM) and poly-(N-isopropyl methacrylamide) (PNIPMAM) which shrink upon application of a temperature trigger and release encapsulated proteins along with the expulsion of water [41,42]. As cross-linker for these nanogels, we used biocompatible dendritic polyglycerol (dPG), which stabilized the nanogels and decreased protein surface adhesion, both important factors for the biodistribution of nanocarriers [[43], [44], [45], [46], [47]].

In a preliminary work we could recently show the feasibility of thermo-responsive nanogels as a stabilizing matrix for the photothermal transducing polymer PANI and we could demonstrate the applicability of the resulting nanogels as PTT agent [16]. For the introduction of PANI we used semi-interpenetration method. Semi-interpenetration is defined as the physical entanglement of a linear polymer in a cross-linked polymeric network and is typically achieved through the formation of one system in presence of the other [48,49]. It emerged as an elegant tool for the inclusion of new capabilities to nanogels through the penetration with functional polymers. The great advantage of the semi-interpenetration method is that the individual properties of both the nanogels and the interpenetrating polymer are retained by their non-covalent linkage. For a potential use of the semi-interpenetrated materials as drug delivery agent, a proper understanding of the interpenetrating process and resulting structure, e.g. the localization of the interpenetrating polymer within the nanogels is important. In the case of stimuli-responsive nanogels, additionally a thorough understanding of structural changes influencing the particles properties under exposure to the stimulus is relevant to determine suitable candidates for an application in vivo.

In the present study, we fabricate thermo-responsive nanogels loaded with the photothermal transducing polymer PPY for combinational photothermal- and chemotherapy along with PA imaging modalities. We prepared thermo-responsive nanogels with different volume phase transition temperatures (VPTTs) by precipitation polymerization using acrylated dPG as cross-linker for PNIPAM, PNIPMAM or copolymers of both and investigated their suitability for a semi-interpenetration with PPY [41]. The photothermal transducing property of PPY endows the nanogels with a dual-response for temperature and NIR light. The generated heat under exposure to NIR can thereby be used as the trigger for the thermo-responsive network as well as for photothermal ablation of cancer. Additionally, the suitability of the prepared nanogels for PA contrast enhancement was assessed using mouse organs as a model. Based on that we developed a new label free method for the establishment of biodistribution profiles using the photothermal response of the particles. Finally, the nanogels are evaluated in vitro and in vivo as drug delivery agent and in combination with photothermal induced ablation of tumor cells. The promising therapeutic outcome encourages further exploration of multifunctional polymer based nanoplatforms for combinational therapy approaches against cancer.