NANOGEL COMPOUND

Abstract

It is provided a nanogel compound comprising a) a nanogel matrix component chosen from the group consisting of poly(N-isopropylacrylamide), poly(N-isopropyl methacrylamide), poly(N,N-diethylacrylamide), poly(N-ethylmethacrylamide), polyglycerol, polyglycerol derivatives and combinations thereof and b) a first or second conjugation component which forms an interpenetrating or semi-interpenetrating network with the nanogel matrix component. Thereby, the first conjugation component is a charged component chosen from the group consisting of acids, methacrylates, methacrylamides, acrylamides and combinations thereof and the second conjugation component is a conjugated polymer chosen from the group consisting of polyaniline, polypyrrole, poly(N(C.sub.1-C.sub.10)alkyl aniline), poly(3,4-ethylenedioxythiophene), 2-ethylhexylcyclopentadithiophene-co-2,1,3-benzothiadiazole, 2-ethylhexylcyclopentadithiophene-co-2,1,3-benzoselenadiazole, and combinations thereof.

Claims

1. A nonogel compound comprising: a nanogel matrix component chosen from the group consisting of poly(N-isopropylacrylamide), poly(N-isopropyl methacrylamide), poly(N,N-diethylacrylamide), poly(N-ethylmethacrylamide), polyglycerol, polyglycerol derivatives and combinations thereof, and a first conjugation component which forms a semi-interpenetrating network with the nanogel matrix component, wherein the first conjugation component is a charged component chosen from the group consisting of acids, methacrylates, methacrylamides, acrylamides and combinations thereof.

2. The nonogel compound according to claim 1, wherein the first conjugation component is chosen from the group consisting of acrylic acid, methacrylic acid, 2-acrylamido-2-methyl-1-propanesulfonic acid, 4-acryloyl amine-4-(carboxyethyl) heptanodioic acid, di(C.sub.1-C.sub.10)alkylamino(C.sub.1-C.sub.10)alkyl methacrylate, N-(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)methacrylamide, N-(3-((4-aminobutyl)amino)propyl)methacrylamide, N-(2-((2-aminoethyl)(methyl)amino)ethyl) methacrylamide, and N-(2-(bis(2-aminoethyl)amino)ethyl)methacrylamide, N-(3-((4-aminobutyl)amino)propyl)acrylamide, N-(2-((2-aminoethyl)(methyl)amino)ethyl)acrylamide, and N-(2-(bis(2-aminoethyl)amino)ethyl)acrylamide and combinations thereof.

3. The nonogel compound according to claim 2, wherein the di(C.sub.1-C.sub.10)alkylamino(C.sub.1-C.sub.10)alkyl methacrylate is dimethylaminomethyl methacrylate, dimethylaminoethyl methacrylate or dimethylaminopropyl methacrylate.

4. The nonogel compound according to claim 1, further comprising a second conjugation component which forms an interpenetrating or semi-interpenetrating network with the nanogel matrix component, wherein the second conjugation component is chosen from the group consisting of polyaniline, polypyrrole, poly(N(C.sub.1-C.sub.10)alkyl aniline), poly(3,4-ethylenedioxythiophene), 2-ethylhexylcyclopentadithiophene-co-2,1,3-benzothiadiazole, 2-ethylhexylcyclopentadithiophene-co-2,1,3-benzoselenadiazole, and combinations thereof.

5. The nonogel compound according to claim 0, wherein the poly(N(C.sub.1-C.sub.10)alkyl aniline) is poly(N-methyl aniline).

6. The nonogel compound according to claim 0, wherein the second conjugation component is polyaniline or polypyrrole.

7. The nonogel compound according to claim 1, wherein the nanogel matrix component comprises dendritic polyglycerol crosslinked by poly(N-isopropylacrylamide).

8. The nonogel compound according to claim 1, wherein the first conjugation component is dimethylaminoethyl methacrylate or 2-acrylamido-2-methyl-1-propanesulfonic acid.

9. (canceled)

10. A medicament comprising a nanogel compound according to claim 0 and a pharmaceutically active substance bound to the nanogel compound.

11. The medicament according to claim 9, wherein the pharmaceutically active substance is chosen from the group consisting of doxorubicin, paclitaxel, methotrexate, cisplatinum, 5-fluorouracil and combinations thereof.

12. A method for producing a nanogel compound according to claim 0, comprising: providing a nanogel matrix component chosen from the group consisting of poly(N-isopropylacrylamide), poly(N-isopropyl methacrylamide), poly(N,N-diethylacrylamide), poly(N-ethylmethacrylamide), polyglycerol, polyglycerol derivatives and combinations thereof, and performing an in situ radical polymerization of a first conjugation component in presence of the nanogel matrix component, the first conjugation component being a charged component chosen from the group consisting of acids, methacrylates, methacrylamides, acrylamides and combinations thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Further aspects and details of the instant invention will be explained with respect to Figures and exemplary embodiments.

[0043] FIG. 1 shows a schematic depiction of the structural behavior of an exemplary embodiment of a nanogel compound.

[0044] FIG. 2A shows the influence of the pH value on the zeta potential and the size of PNIPAM-dPG/DMAEM.

[0045] FIG. 2B shows the influence of the temperature on the zeta potential of PNIPAM-dPG/DMAEM.

[0046] FIG. 2C shows the influence of the pH value on the zeta potential and the size of PNIPAM-dPG/AMPS.

[0047] FIG. 2D shows the influence of the temperature on the zeta potential of PNIPAM-dPG/AMPS.

[0048] FIG. 3 shows the cumulative release of DOXO from PNIPAM-dPG/DMAEM and PNIPAM-dPG/AMPS at 25 and 42 C. and at pH 7.4.

[0049] FIG. 4 shows a plot of the cytotoxicity profile of PNIPAM-dPG/DMAEM and PNIPAM-dPG/AMPS determined by an MTT assay.

[0050] FIG. 5A shows a plot of the cytotoxicity profile of DOXO-loaded PNIPAM-dPG/DMAEM and PNIPAM-dPG/AMPS and free DOXO determined by an MTT assay in HeLa cells.

[0051] FIG. 5B shows a plot of the cytotoxicity profile of DOXO-loaded PNIPAM-dPG/DMAEM and PNIPAM-dPG/AMPS and free DOXO determined by an MTT assay in resistant cell lines (KB-V1).

[0052] FIG. 6 shows the in vivo evaluation of bare and DOXO-loaded PNIPAM-dPG, PNIPAM-dPG/AMPS, PNIPAM-dPG/DMAEM, and of free DOXO.

[0053] FIG. 7 shows a schematic depiction of the synthesize and of the structural behavior of a nanogel compound not comprising a charged conjugation component.

[0054] FIG. 8A shows the thermoresponsive behavior of PNIPAM-dPG/PANI measured by Dynamic Light Scattering (DLS).

[0055] FIG. 8B shows a micrograph obtained by Atomic Force Microscopy (AFM) of PNIPAM-dPG/PANI.

[0056] FIG. 8C shows a micrograph obtained by Atomic Force Microscopy (AFM) of PNIPAM-dPG/PPY.

[0057] FIG. 9 shows the change of temperature over time of irradiation for dispersions of PNIPAM-dPG/PANI at different concentrations.

[0058] FIG. 10A shows the cytotoxicity of PNIPAM and PNIPAM-dPG/PANI on A2780 cells as determined by an MTT assay.

[0059] FIG. 10B shows the PNIPAM-dPG/PANI concentration in culture medium of A2780 cells as determined by absorbance measurement over time.

[0060] FIG. 11 shows the relative viabilities of A2780 cells, treated with PNIPAM-dPG/PANI or untreated, irradiated with NIR laser or heated to 42 C.

[0061] FIG. 12 shows the tumor growth inhibition (%) of tumors in female nude mice treated with PBS, PNIPAM-dPG, PNIPAM-dPG/PANI, PNIPAM-dPG/PPY with and without irradiation.

[0062] FIG. 13 shows the cumulative release of DOXO from PNIPAM-dPG/PANI at pH 7.4 with and without NIR irradiation.

[0063] FIG. 14 shows the temperature change of a cell cluster of 1 million cells, incubated with drug-loaded semi interpenetrated nanogels, during irradiation with NIR laser.

[0064] FIG. 15 shows the cell viability of irradiated or non-irradiated DOXO-loaded PNIPAM-dPG/PANI or PNIPAM-dPG/PPY 24 h after treatment.

[0065] FIG. 16 shows MTX release from PNIPAM-dPG/PPY nanogels above or below the transition temperature, or after NIR laser irradiation.

DETAILED DESCRIPTION

[0066] FIG. 1 shows the structural behavior of a thermoresponsive nanogel compound 1 based on poly(N-isopropylacrylamide) (PNIPAM) 2 and dendritic polyglycerol (dPG) 3 which is semi interpenetrated (s-IPN) with a charged polymer, namely, dimethylaminoethyl methacrylate (DMAEM) 4 as first conjugation component. Alternatively, 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) or another charged polymer could be used as first conjugation component. For sake of clarity, only some of the respective molecules of the individual components in the depiction of FIG. 1 bear the corresponding numeral reference. Upon a temperature increase, the nanogel compound 1 shrinks, i.e. its size is reduced. Thereby, the positive charges of DMAEM 4 are exposed to the outside of the core of the nanogel compound 1. If AMPS was used as first conjugation component, its negative charges would be exposed to the outside of the core of the nanogel compound. Thus, the temperature increase triggers an exposure of the charge of the first conjugation component.

[0067] The charge density of the nanogel compound can be controlled by the ratio between charged monomer (first conjugation component) and the nanogel matrix component as well as by the time of polymerization of the charged monomer. The successful semi-interpenetration of DMAEM or AMPS was proven by Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (.sup.1H NMR).

[0068] After the semi-interpenetration of the nanogel matrix component by the first conjugation component, the zeta potential of the network changes. When the nanogel matrix component is semi-interpenetrated by a cationic or anionic compound, the zeta potential is more positive or negative, respectively. By increasing the percentage of the charged compound in the network or the time of reaction of the charged compound, the zeta potential increases as well.

[0069] Interestingly, the transition temperatures of the nanogel compounds do not change after the semi-interpenetration with the first conjugation component. This behavior is opposite to the one observed when the first conjugation component is co-polymerized into the network of the nanogel matrix component..sup.19

[0070] Two different behaviors can be obtained depending on the chosen first conjugation component. As a general rule, the size of the nanogel compound increases with the charge of the network. Thus, when increasing the pH, the positively charged PNIPAM-dPG/DMAEM nanogel compound shows a decrease of size. To the contrary, the negatively charged PNIPAM-dPG/AMPS nanogel compound shows an increase in size when the pH value is increased. This is shown in FIGS. 2A (PNIPAM-dPG/DMAEM) and 2C (PNIPAM-dPG/AMPS). Thereby, the circles in FIGS. 2A and 2C relate to the size (right y-axis), whereas the squares relate to the zeta potential (left y-axis).

[0071] In the case of PNIPAM-dPG/DMAEM NGs the size decreased from 430 nm at pH 3 to 175 nm at pH 10 with a change in zeta potential from 20 to 2. The zeta potential of PNIPAM-dPG/AMPS NGs changed from 3 to 3 with the increase of pH from 2 to 10, followed by an increase of size from 150 nm to 400 nm. Moreover, after collapse of the nanogel compound, the zeta potential of the network increased. This is shown in FIGS. 2B (PNIPAM-dPG/DMAEM) and 2D (PNIPAM-dPG/AMPS). This can be explained by the exposition of the charged first conjugation component during the collapse of the thermoresponsive nanogel compound. This behavior can be exploited in different fields and applications, for example to encapsulate or release a cargo on demand.

[0072] In order to use these new nanogel compounds in drug delivery applications, the encapsulation of the anti-cancer drug doxorubicin (DOXO) was studied. The results of the encapsulation study are shown in Table 1. PNIPAM-dPG/AMPS encapsulates the double amount of DOXO compared to PNIPAM-dPG/DMAEM due to the electrostatic interaction. As a comparison PNIPAM/dPG without charges was also studied.

TABLE-US-00001 TABLE 1 Loading capacity (L.C., in %) and loading efficiency (L.E., in %) of DOXO in PNIPAM, PNIPAM-dPG/AMPS, and PNIPAM-dPG/DMAEM. Nanogel compound % L.C. % L.E. PNIPAM/dPG (control) 6.48 13.25 PNIPAM-dPG/AMPS 8.97 17.50 PNIPAM-dPG/DMAEM 4.30 9.00

[0073] After the encapsulation, the release of DOXO was studied in vitro. For each nanogel compound, two experiments were performed below (25 C.) and above (42 C.) the transition temperature in order to see if the release is triggered by the collapse of the nanogel compound. The results are shown in FIG. 3.

[0074] When an electrostatic interaction is present the release is slower than without electrostatic interaction, while for all nanogel compounds the release is faster at 42 C. than at 25 C. showing a temperature-dependent release.

[0075] The in vitro toxicity and cellular uptake in HeLa cell lines were studied for further utilization of these materials in biomedical applications. The results are shown in FIG. 4. None of the tested nanogel compounds showed an effect on this cell line up to concentration of 1 mg mL.sup.1, indicating their very good biocompatibility.

[0076] In order to use the nanogel compounds for anticancer therapy, the cytotoxicity of the DOXO-loaded nanogel compounds compared with free DOXO was studied in two different cell lines: sensitive-type HeLa cells and DOXO resistant cell line KB-V1. The results are shown in FIGS. 5A and 5B. Thereby, squares denote PNIPAM-dPG/AMPS, circles denote PNIPAM-dPG/DMAEM, and triangles denote free DOXO.

[0077] Briefly, with the sensitive-type HeLa cells (FIG. 5A) the DOXO-dose dependent cytotoxicity curve of the nanogel compounds was similar to free DOXO. In contrast, with the cell resistant line (KB-V1) the external concentration of free DOXO of up to 10 M had no effect on cytotoxicity, which is typical for resistant cell lines, indicating that the active internalization of DOXO is essential for toxicity (FIG. 5B). The dose-response curve of the DOXO-loaded nanogel compounds resembled very closely that one obtained for the sensitive cells. This demonstrates the efficiency of these nanogel compounds as carriers for drugs to overcome drug resistance in tumor cell lines.

[0078] To further prove the efficacy of these carriers, in vivo tumor growth inhibition experiments were carried out. In order to test the potency of the DOXO-loaded nanogel compounds to overcome anthracycline resistance in vivo, nude mice were injected with HeLa-derived xervic carcinoma Adriamycin-resistant MaTu-ADR and treated twice with the DOXO-loaded nanogel compounds using phosphate-buffered saline (PBS) and bare (unloaded) nanogel compounds as negative control and compared with free DOXO.

[0079] The results are shown in FIG. 6. It can be clearly seen that the group treated with DOXO-loaded PNIPAM-dPG/AMPS could effectively overcome the DOXO resistance of the cells and inhibited the tumor growth after 32 days of treatment. The efficiency of this carrier is likely due to the strong electrostatic interaction between the DOXO and the charges of the nanogel compound which enable the prolonged controlled release of the drug over the time, which provoked a low but constant DOXO concentration inside the cell which is not being detected by the efflux pumps of the cells.

[0080] In summary, novel drug carriers were developed by semi-interpenetration of a charged compound inside a thermoresponsive nanogel matrix component. These novel nanogel compounds increase the superficial charge on demand, after an increase in the temperature. The charge of the thermoresponsive nanogel compound can be finely tuned by the chosen charged component, the ratio of charged component to nanogel matrix component, and the polymerization time of the charged component inside the nanogel matrix component.

[0081] DOXO, an anti-cancer drug was encapsulated and efficiently released triggered by an increase in the temperature of the surrounding medium. While the encapsulation of the drug is increased due to the electrostatic interactions with the novel nanogel compound, the release is slower when this interaction is present. On the other hand, when electrostatic repulsion is observed, the release of the drug is much faster. Low cytotoxicity profile and good internalization of the nanogel compounds by tumor cells were shown. This is important for a use of the nanogel compounds as drug carrier. In comparison with free DOXO, the DOXO-loaded nanogel compounds have been proven to efficiently overcome the pump efflux of the drug resistant cells, showing a high cytotoxicity. More importantly, DOXO-loaded PNIPAM-dPG/AMPS has been proven to efficiently overcome the resistance of cells in vivo.

[0082] Thus, suited applications of the described nanogel compounds are controlled drug/protein delivery and anticancer therapy.

[0083] In further experiments, nanogel compounds comprising a semi-interpenetrating second conjugation component which is not a charged component have been prepared. No first conjugation component was used. These nanogel compounds are also very well suited as drug carriers, in particular for anticancer drugs.

[0084] With reference to FIG. 7, a thermoresponsive nanogel comprising a nanogel matrix 2, 3 which is semi-interpenetrated (s-IPN) with a conjugated polymer 5, namely, polyaniline (PANI) or polypyrrole (PPY) has been synthesized. This composite serves as nanogel compound. The conjugated polymer 5 (second conjugation component) generates heat after being irradiated by near infrared (NIR) radiation, inducing a local hyperthermia. This is also schematically depicted in FIG. 7. The nanogel compound, because of being thermosensitive, undergoes a change in conformation that triggers different mechanisms like shrinkage of the nanogel compound, aggregation of the nanogel compound, drug 6 release from the nanogel compound (if it was previously loaded with this drug 6), uptake of the nanogel compound into a cell and/or a tissue as well as accumulation in a specific compartment, etc.

[0085] The thermoresponsive nanogel compound was synthesized based on the work of Cuggino et al..sup.14 Poly(N-isopropylacrylamide) (PNIPAM) 2 was used as thermoresponsive polymer (a first nanogel matrix component) and vinyl functionalized dendritic polyglycerol (dPG) 3 was used as macro-crosslinker (a second nanogel matrix component). The synthesis of the nanogel compound (PNIPAM-dPG/PANI or PNIPAM-dPG/PPY) was done by an in situ redox polymerization of aniline or pyrrole in the presence of the thermoresponsive nanogel matrix component.

[0086] A stable PNIPAM-dPG/PANI or PNIPAM-dPG/PPY dispersion with reproducible size and thermoresponsive properties was obtained using 0.1 M of aniline or pyrrole and 30 min of polymerization. According micrographs obtained by atomic force microscopy are depicted in FIGS. 8B and 8C. FIG. 8A shows the thermoresponsive behavior of PNIPAM-dPG/PANI. Its transition temperature is at approximately 32 C. Heating PNIPAM-dPG/PANI above its transition temperature leads to a significant decrease in size, i.e. to shrinkage or a collapse of PNIPAM-dPG/PANI.

[0087] A suited application of this nanogel compound is hyperthermia therapy. This was probed by studying the increase of the temperature of the nanogel compound under NIR radiation. As can be seen in FIG. 9, the increase in temperature of a dispersion of the nanogel compound in a suited medium is dependent on the concentration of the nanogel compound. Thereby, the increase in temperature is proportional to the concentration of nanogel compound in the dispersion. When only water is irradiated, the temperature increases by 3 C. If 20 g mL.sup.1 of PNIPAM-dPG/PANI in water is irradiated under the same conditions, the temperature increases already by 8 C., which is enough for mild hyperthermia conditions..sup.20

[0088] The cytotoxicity of the nanogel compound on A2780 ovarian cancer cells was studied by MTT assay (MTT stands for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The results depicted in FIGS. 10A and 10B.

[0089] The cells which were incubated with PNIPAM-dPG/PANI or PNIPAM-dPG alone, show high viability over 50% up to a concentration of 1 mg mL.sup.1 (FIG. 10A). Uptake of PNIPAM-dPG/PANI into cells was monitored indirectly by measuring the concentration in cell culture supernatant (FIG. 10B). After 64 h, the concentration was about 0.03 mg mL.sup.1 less than initially incubated. Thus, this amount was taken up by the cells. Additionally, it was observed that the cells were dark colored after trypsinization and washing with PBS or media. The color remained after repeated trypsinization, reculturing, and washing. This confirms that the nanogel compound was located inside the cells.

[0090] For the study of the hyperthermia therapy in vitro, cells were released from a cell culture flask, washed and centrifuged into PCR tubes which allow for rapid transmission of heat through the walls of the tubes. In this way, the approximate temperature of the cell suspension or cell pellet could be monitored. The culture medium was removed leaving a small ball of approximately 1 million cells that had about the volume of a 5 L drop. This cluster of cells, which may resemble a small tumor tissue, was irradiated with a NIR laser (785 nm) under constant monitoring of a thermal camera to ensure the temperature did not exceed 42 C.+/1 C. As a control for the effect of hyperthermia alone, equally treated samples were incubated in a water bath at 42 C. Irradiation or heating was performed one time for 5 min or three times for 5 min each with PNIPAM-dPG/PANI incubated cells as well as with untreated cells. Viabilities were measured after 24 h with MTT assay. The results are shown in FIG. 11.

[0091] A2780 cells which had taken up PNIPAM-dPG/PANI heated up during irradiation with a NIR laser and showed a markedly reduced viability after a single treatment for 5 min, as compared to 80% viability of untreated control cells which were irradiated for the same time. Four irradiation cycles resulted in a complete loss of cell viability of PNIPAM-dPG/PANI treated cells, whereas the control cells again were not compromised. The effect of hyperthermia for 5 min alone was not measurable. Only a decrease in viability to about 60% could be observed if the hyperthermia is repeated four times. To control for the stress of centrifuging and standing as a cell cluster for a prolonged period of time outside of the incubator, another cell sample was treated in the same way but not heated or irradiated. These control cells showed no reduced viability, comparable to the untreated cells in the other series.

[0092] The cytotoxicity of the nanogel compound was also studied in vivo by two different essays. First a single treatment with doses from 10 to 100 mg kg.sup.1 showed that mice tolerated up to 100 mg kg.sup.1 of PNIPAM-dPG/PANI or PNIPAM-dPG/PPY without any significant sign of toxicity or drug-related body weight loss. In a second step, three mice were treated with the maximum tolerated dose (MTD) found in the single treatment, 100 mg kg.sup.1 once per day at 5 consecutive days. Mice were observed for a follow-up period of 18 days. No significant toxic effect was observed during that period. A dark blue color was observed in the tails, liver area, and blood vessel system in the abdomen.

[0093] Body weight was slightly reduced to 97.2% at day 11, but mice recovered very fast from this effect.

[0094] The therapeutic effect in vivo was also studied. A2708 tumor transplants were obtained from tumors, cut into small pieces (222 mm) and transplanted subcutaneously (s.c.) into female nude mice (Taconic) at day 0. Mice were classified at day 14 into 10 groups with 5 mice each. Mean tumor volume at this time was 0.086-0.088 cm.sup.3/group. Mice were intratumorally (i.t.) or intravenously (i.v.) injected with PNIPAM-dPG/PANI or PNIPAM-dPG/PPY using PBS and PNIPAM-dPG as controls. At indicated times, mice were narcotized and tumors exposed to NIR laser light at distance of about 5 cm for 5 min at a maximum radiation power of 500 mW. Temperature profiles of each mouse were taken during each exposure. Two to three times a week, body weight and tumor volume (TV) was measured. The experiment was finished at day 39. FIG. 12 shows the obtained results.

[0095] As can be seen in FIG. 12, there is no or only a weak inhibitory effect of the nanogel compounds without irradiation as well as with the control PNIPAM-dPG with NIR. Contrary to that, the injection of PNIPAM-dPG/PANI followed by treatment with NIR light resulted in a tumor growth inhibition by 58% (tumor volume changetreated to control (T/C)=42%). That was still improved if mice were treated i.t. with PNIPAM-dPG/PPY and immediately exposed to NIR laser light. In this way the best inhibition of 86% (T/C=14%) was obtained.

[0096] Moreover, it is possible to encapsulate anticancer drugs inside the nanogel compounds in order to obtain a synergetic therapy combining photothermal with chemotherapy. To investigate the potential of the described nanogel compounds as NIR-sensitive drug delivery system, the encapsulation of DOXO and the release behavior dependent on temperature and NIR irradiation was studied.

[0097] The encapsulation results dependent on the DOXO feed for PNIPAM-dPG/PANI are given in Table 2. The higher the initial feed of DOXO, the higher the loading capacity of PNIPAM-dPG/PANI. Specifically, the loading capacity (LC) increases from 0.9% at 2.8 wt % DOXO feed to 13.2% at a DOXO feed of 53 wt %. The comparison of the nanogel compounds with bare PNIPAM-dPG shows almost a doubling of DOXO loading inside the NGs at 50 wt % DOXO feed. This indicates a strong impact of PANT on the encapsulation process since conjugated polymers can stabilize DOXO inside the nanogel compound by it-it-stacking.

TABLE-US-00002 TABLE 2 Loading capacity (LC, in %) and loading efficiency (LE, in %) of DOXO in PNIPAM-dPG and PNIPAM-dPG/PANI dependent on initial DOXO feed per mg of nanogel compound [wt %]. PNIPAM-dPG/PANI PNIPAM-dPG DOXO feed (wt %) 2.8 4.7 9.3 53 50 % LC 0.9 2.1 3.1 13.2 6.5 % LE 34 46 33 25 13

[0098] To demonstrate the ability for NIR-triggered drug release from PNIPAM-dPG/PANI, release behavior upon NIR laser irradiation was investigated. Therefore, the PNIPAM-dPG/PANI with encapsulated DOXO was exposed to NIR light before a dialysis was started and after 2 and 4 h during dialysis by placing the dialysis tube in front of a NIR-Laser (=785 nm, 500 mW) for 5 min.

[0099] The results are presented in FIG. 13. By comparing with the release without laser irradiation it can be clearly seen that an increase of the free DOXO could be achieved upon NIR irradiation. An abrupt rise of DOXO release directly after the irradiation is visible which can be correlated to a NIR-induced heat production by PANI leading to shrinkage of the nanogel compound.

[0100] By combining the controlled delivery of DOXO, triggered by NIR, with the photothermal ablation of the cancer cells it is expected to see a synergistic tumor growth inhibition both in vitro and in vivo. After ablation of the cancer cells by NIR absorption, the nanogel compounds will slowly continue releasing DOXO, thus inhibiting the tumor growth.

[0101] In a further experiment, PNIPAM-dPG/PANI and PNIPAM-dPG/PPY were loaded with DOXO using the thermoresponsive properties of the nanogels. The loading capacity was found to be 13-14%, which is about twice the amount that can be loaded to non-semiinterpenetrated PNIPAM-dPG NGs.

[0102] When incubated with HeLa cells at 0.1 mg/ml overnight, PNIPAM-dPG/PANI-DOX and PNIPAM-dPG/PPY-DOX were uptaken, causing a dark color in the cells. Cells were washed and irradiated as a cluster of 1 million cells with a NIR laser for 5 minutes. During irradiation, the temperature was increased by about 42 C. for PNIPAM-dPG/PANI-DOX and by about 60 C. for PNIPAM-dPG/PPY-DOX.

[0103] FIG. 14 shows this temperature change of the examined cell cluster. Non-loaded cells do not show any temperature increase due to NIR irradiation.

[0104] To prove that this temperature increase will cause cell ablation in addition to the antiproliferative effect of DOXO, cells were reseeded after treatment and their viabilities were determined using MTT assay after 24 h.

[0105] For PNIPAM-dPG/PANI-DOXO, cell viability was reduced to about 50% in non-irradiated cells, showing the cytotoxic effect of the drug alone. However, the cell viability was reduced to almost zero for the irradiated cells, showing the synergistic and combinatory effect of the two treatments (DOXO plus NIR irradiation). Similar results were obtained for PNIPAM-dPG/PPY-DOXO, with a less pronounced effect of DOXO alone, indicating a different drug release kinetics.

[0106] The according results are shown in FIG. 15.

[0107] PNIPAM-dPG/PPY nanogels were also loaded with another drug, namely the folate analogue Methotrexate (MTX), using the thermoresponsive properties of the nanogel. The loading capacity was found to be about 18%.

[0108] The drug release profiles show that MTX release was strongly enhanced upon NIR laser irradiation. In contrast, if a small temperature trigger was provided leading to a temperature increase above the transition temperature of the nanogels, MTX release could not be increased significantly. This clearly shows that NIR treatment leads to a much more prominent temperature increase resulting in a stronger destabilization of the nanogels than a simple temperature increase above the transition temperature of the nanogels.

[0109] In summary, the inventors have formulated NIR sensitive organic nanoparticles based on PNIPAM-dPG/PANI or PNIPAM-dPG/PPY as a novel photothermal agent and used for highly effective in vitro hyperthermia treatment of cancer cells. By semi interpenetrating conjugated polymers in a thermoresponsive polymeric nanogel matrix component, a nanocomposite (nanogel compound) with excellent compatibility in physiological environments was obtained. This nanogel compound provides the opportunity to load the nanogel compound with drugs, such as anticancer drugs, as well as to attach targeting moieties to the nanogel matrix component, such as dPG.

[0110] Thus, these nanogel compounds are suited tools for cancer therapy. Moreover, the studies in vivo showed that mice tolerated up to 100 mg/kg of PNIPAM-dPG/PANI or PNIPAM-dPG/PPY, given at 5 consecutive days (accumulated dose: 500 mg kg.sup.1) without any significant sign of toxicity.

[0111] And moreover, the therapeutic effect study indicated that the combination of systemic treatment with PNIPAM-dPG/PANI or PNIPAM-dPG/PPY and NIR exposure results in a higher sensitivity of the tumors for treatment with anticancer drugs if compared to the controls alone or with NIR.

[0112] Furthermore, the possibility of encapsulation and controlled slow release of DOXO after NIR irradiation was demonstrated. Combining these features, the described nanogel compounds can be applied in a combinatorial chemo- and photothermal therapy for anticancer treatment. Thus, suited applications of the described nanogel compounds are hyperthermia for cancer therapy, drug delivery triggered by NIR and combined chemo and photothermal cancer therapy.

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