Poly (N-isopropylacrylamide)-functionalized dendrimer as a thermosensitive nanoplatform for delivering malloapelta B against HepG2 cancer cell proliferation

Poly(N-isopropylacrylamide) maleimide and dimethyl formamide were purchased by Sigma. PAMAM dendrimer was synthesized by Department of Materials and Pharmacy Chemistry (Institute of Applied Materials Science) following the Tomalia process [5, 20]. Mall B was isolated from the leaves of M. apelta conducted by Institute of Marine Biochemistry (IMBC-VAST). Regenerated cellulose MWCO 3500–5000 Da and 14 000 Da dialysis bags were from Spectrum Laboratories Inc. All other chemicals and solvent were used without further purification.

PNIPAM-g-PAMAM G3.5 was synthesized via two steps of Michael reaction. Briefly, PAMAM G3.0 (0.045 mmol) and maleimide-terminated PNIPAM (0.315 mmol) were separately dissolved in 20 ml of DMF, and then, PAMAM G3.0 solution was added drop-wise under stirring condition. The reaction was conducted at a room temperature in 48 hours under N₂ condition. After that, excess methyl acrylate (0.3 mmol) was slowly added into the mixture to convert all unreactive aminegroups. The reaction was continuously kept at the room temperature stirring condition in 96 hours with N₂. Then, it was dialyzed by regenerated cellulose MWCO 12 000–14 000 Da dialysis bags in methanol during 4 d and removed solvents from the sample via rotary evaporation (figure 2).The copolymer was characterized by ¹H NMR and TEM, DLS and UV–Vis measurement.

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To record thermosensitive property of the grafted copolymer, the optical absorbance of PNIPAM and PNIPAM-g-PAMAM derivative solutions in distilled water (1 mg ml⁻¹) at various temperatures was measured at 293 nm photolab^®6600 UV–Vis spectrometer. The sample cells were thermo-stated at different temperatures from 30 °C to 35 °C prior to measurements. The LCST values of the sample solutions were defined as the temperature showing an optical transmittance of 50%.

20 mg of Mall B and PNIPAM-g-PAMAM G3.5 was dissolved in mixture of 20 ml polysorbabe 80 (0.0625%, tween 80) and 20 ml of ethanol then sonicated for 15 min. After that, Mall B-containing solution was vapor rotated and free-dried to attain the Mall B-loaded copolymer. The Mall B-loaded PNIPAM-g-PAMAM G3.5 solution was dispersed again in DI water at 25 °C, warmed up to 37 °C and added into membrane dialysis for indirectly evaluating Mall B loading and releasing efficiencies by UV–Vis method with UV–Vis 6600 spectrophotometer. The evaluation was triplicated. The drug loading (DL %) and entrapment efficiency (EE %) of malloapelta B in thermosensitive dendrimer were calculated from the following equations [36]

$$\begin{eqnarray}&&\begin{array}{*{20}{l}}\text{DL}\left(\%\right)=\frac{\,\text{Mass} ~\text{of} ~\text{loaded} ~\text{malloapelta} ~\text{B}}{\left(\text{Mass} ~\text{of} ~\text{loaded} ~\text{malloapelta} ~\text{B} ~ +\text{Mass} ~\text{of} ~\text{polymer} ~ \right)}\\ \quad \quad \ \ \;\,\,\,\times 100,\end{array}\end{eqnarray}$$

$$\begin{eqnarray}&&\begin{array}{*{20}{l}}\text{EE}\left(\%\right)=\frac{\text{Total} ~\text{malloapelta} ~\text{B} ~\text{amount} ~ -\,\text{free} ~\text{malloapelta} ~\text{B} ~\text{amount}}{\text{Initial} ~\text{mass} ~\text{of} ~\text{malloapelta} ~\text{B} ~}\\ \qquad \,\,\,\quad \,\,\times 100.\end{array}\end{eqnarray}$$

The Mall B-loaded colloidal copolymer solution was added to the dialysis bag 3500 Da and dialyzed with 12 ml of phosphate buffer saline (PBS) solutions at pH 7.4 at 37 °C. At the predetermined time interval, 1 ml of dialyzed solution was drawn to determine Mall B and replaced with an equivalent volume of PBS into each tube. Mall B release was determined by the UV–Vis method with the absorption wavelength at 264 nm.

The experiment was conducted via sulfordodamine B (SRB) colorimetric assay at Faculty of Biology—University of Science, Vietnam National University in Ho Chi Minh City. Similarly, PNIPAM-g-PAMAM G3.5, free Mall B, and Mall B loaded in PNIPAM-g-PAMAM G3.5 were optimized at the screening to test the inhibition capability of cell growth. The HepG2 cell line preserved in liquid nitrogen was thawed to culture them to 4th generation. Then, HepG2 cells were seeded in 96 well plates (10⁴ cells/well) and allowed 24 h for cell growth 4 in culture medium with 5% CO₂ atmosphere at 37 °C. Then, the medium was replaced by the tested samples with a predetermined concentration and incubated for 48 h later. The culture medium were divided into 2 separate medium, which one considered as 'negative control' while another was 'blank sample' containing samples but without cells. After being incubated, the cells were fixed with 50% (wt/vol) trichloroacetic acid and stained with 0.2% (wt/vol) SRB for 20 min. The samples were washed repeatedly (5 times) and clearly with 1% (vol/vol) acetic acid. Finally, the protein-bound dye was dissolved in 10 mM tris-base solution to determine the optical density (OD) with the absorption wavelength at 492 nm and 620 nm. Based on the OD results of control, blank and sample the growth inhibition values were calculated [37, 38].

In figure 3 the proton peak of the PNIPAM-g-PAMAM G3.5 copolymer was clearly indicated that there was a co-existence of PAMAM G3.5 with several typical resonance signals such as peak (d) at 3.73–3.78 ppm [–COOCH₃], (e) at 3.26–3.36 ppm [–CONHCH₂CH₂N], (a) at 2.57–2.63 ppm [–CH₂CH₂N] and the polymer PNIPAM signals as –NHCOCH(CH₂)₂ (peak (i)), –CH₂-(peak (h)), CH(CH₃)₂NHCO-(peak (l)). Apparently, it was indicated that thermosensitive NiPAM polymer grafted into PAMAM and methylacrylate was reacted to-NH₂ groups of PNIPAM-g-PAMAM G3.0. Hence, it was seen that appearance of the conjugated methyl acrylate and PAMAM dendrimer PNIPAM-g-PNIPAM protons.

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Furthermore, morphologies of the thermosensitive nanocarriers were observed by transmission electron microscopy (TEM) as shown in figure 4 with the size of the PNIPAM-g-PAMAM G3.5, in the range of 70–120 nm at 37 °C, which was potential in application of the drug delivery nanocarriers.

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Remarkably, dynamic light scattering (DLS) analysis demonstrated that the hydrodynamic size of nanocarriers PNIPAM-g-PAMAM G3.5 was approximately 146.8 nm at 37 °C (figure 5(a)). Besides, the entrapment of drug into nanocarriers could be enhanced with the size up to 194.5 nm with the same temperature (figure 5(b)).

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It was shown the temperature made a profound impact on the light transmittance of the PNIPAM-g-PAMAM with different derivatives at 293 nm in distilled water (1 mg mL⁻¹). As in figure 6, the PNIPAM-g-PAMAMs solutions were almost transparent approximately 100% when the temperature was controlled lower 31 °C. However, there was a significant decrease in the transmittance of solutions, in which a transition turned into opacity happened while temperature heated up. In comparison with original PNIPAM, the grafted copolymer showed a higher hydrophilic structure due to graft of PAMAM dendrimer. However, the dendrimer-based copolymer also converted into the micelles at temperature ranging from 34 to 36 °C which could offer a great potential for loading hydrophobic drugs.

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As Mall B being highly hydrophobic active compound, it could not be done in water and thermosensitive PNIPAM-g-PAMAM G3.5 was therefore applied to encapsulation in order to improve its poor solubility. In comparison with thermosensitive PNIPAM both the PNIPAM-g-PAMAM-based nanocarriers exhibited a higher loading (DL  =  59.93  ±  0.19%) and entrapment efficiency (EE  =  89.98  ±  2.06%) while it was achieved 52.54  ±  0.45% and 66.45  ±  2.78%, respectively in the PNIPAM as shown in figure 7. The results could indicate that interaction of the hydrophobic compound and polymer platform as well a higher cavity space of the dendrimer-based nanocarrier resulting in significantly enhancing drug encapsulation.

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Mall B was used as a hydrophobic drug model to investigate the release ability of the bioactive compound from the thermosensitive nanocarriers. The drug tracked down from nanocarriers was slower and more stable in the plasma environment (pH 7.4) whilst the PNIPAM-g-PAMAM G3.5 speeded the drug release up in mildly acidic pH  =  5.5. In figure 8, a similar phenomenon was happened to the temperature-responsive polymer PNIPAM with a slightly disparity. Under the alkaline (pH 7.4) environment of plasma, the accumulative Mall B release from the dendritic nanocarrier reached at 8.29  ±  0.4% during the first twenty hours, then there was a steady increase in amount of drug into 12.51  ±  0.53% after 54 h. Furthermore, the PNIPAM release trend was under a similarity to PNIPAM-g-PAMAM G3.5 during the same condition, which achieved 11.49  ±  0.93% and 16.68  ±  0.56% of accumulative drug, respectively, after the first day and much more than 2 d.

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Whereas, in the acidic (pH  =  5.5) environment, PNIPAM and PNIPAM-g-PAMAM G3.5 bore a dissimilarity to those in the neutral. Mall B encapsulated in PNIPAM was achieved the first 24 h with accumulative release rate of approximately 13.33  ±  0.19% then took an upturn to 21.81  ±  0.74% until 54 h, whilst, the PNIPAM-g-PAMAM G3.5 could release 18.53  ±  1.02% and speeded up to 28.71  ±  1.02% of accumulative drug during the similar period. The result is reasonable owning that the nanostructure of thermosensite denrimer nanostructure is swellable due to protonization of amine groups in acidic medium resulting into a higher amount of released drug. Hence, it was promising in the reduction of side-effects of the drug in the blood plasma while facilitating the drug release in the tumor site with acidic medium in control.

HepG2 human liver cancer cell was used to evaluate cytotoxic behavior of thermo-sensitive nanogel PNIPAM-g-PAMAM G3.5, free Mall B and Mall B loaded PNIPAM-g-PAMAM G3.5 using sulforhodamine B dye molecule. The result indicated that PNIPAM-g-PAMAM G3.5 was relatively non-cytotoxic at the experimental condition; merely 6.21  ±  1.87% of the cell was inhibited growth at screening concentration (100 µg ml⁻¹). Whilst, the free drug and drug-encapsulated nanogel exhibited a highly anti-proliferative activity on the cancer cells at the small concentration, in which 50 µg ml⁻¹ of free Mall B reduced 95.57  ±  0.75% of cell growth and 44.75 µg ml⁻¹ of PNIPAM-g-PAMAM G3.5-Mall B induced 97.58  ±  0.83% cell expansion to inhibitory (figure 9).

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Moreover, the free Mall B drug and drug-loaded nanocarriers could inhibit significantly 50% of the HepG2 cancer cell proliferation at 11.32  ±  0.61 µg ml⁻¹ and 20  ±  0.91 µg ml⁻¹, respectively (figure 10). Notably, Mall B-loaded PNIPAM-g-PAMAM G3.5 could exhibit significantly cell growth because amount of Mall B released out of the nanocarrier approximately 20% of loaded amount at point time 48 h (regarding to release profile in figure 8). The result may offers a great potential of the Mall B-loaded PNIPAM-g-PAMAM G3.5 nanocarrierfor inhibiting cancer cell growth in tumor.

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