3.1 Synthesis and characterization of PAMAM dendrimers
The PAMAM dendrimers of various generations (G0.5, G1.0, G1.5, G2.0 and G2.5) are synthesized using the Michael addition method as described in the previous section. The PAMAM dendrimers with ammonia as the core molecule possess an ester group (R-COO-R) and amine (R-CO-NH2) group as terminal surfaces in the successive half and full generation. The various generation dendrimers were characterized by UV–vis spectroscopy, FTIR spectroscopy, AFM and zeta potential. The UV–vis spectroscopy of all generations of PAMAM dendrimers shows a characteristic absorption at 275 to 290 nm (Figure S1). The FTIR spectra of the half generation (G0.5/G1.5/G2.5) shows a characteristic –C = O stretching vibrations around 1,729 cm−1 due to the presence of a free ester (C = O) group in the end surface (Kolhe et al. 2003). In addition to this, a band at 1,000 cm−1 to 1,200 cm−1 also appeared, which is assigned to the –C–O stretching mode [Fig. 3, 2.5 PAMAM]. The FTIR spectrum of the full generation dendrimers shows characteristic N–H stretching vibrations around 1,620–1,650 cm−1 because of the reaction of ester with amine making it as an amine end (Figure S2).
From the zeta potential values of various generations, it is possible to evaluate the nature of the surface groups present in the dendrimers (Tomalia et al. 2007). The half generation PAMAM dendrimers are electrically neutral and show positive potential values. The full generation PAMAM dendrimers are electronegative and they possess negative potential values. The zeta potential data also confirmed the presence of the charged/neutral terminals at the surfaces. The zeta potential values of all the PAMAM dendrimers of various generations are given in Table 1. The AFM images of the 2.5 G PAMAM dendrimer (2D and 3D) recorded in noncontact mode is given as Fig. 1(a) and (b) respectively. From the image, it is clear that most of the particles appeared spherical in shape with particle size around 20 nm.
3.2 Dendrimer–Rose Bengal interaction
The dendritic cavity present in the dendrimer molecules enables them to hold the guest molecule (drug). Such host–guest interactions can be successfully followed spectrophotometrically. UV–visible absorption peak of G2.5 dendrimer and the RB drug in methanol is shown as in Fig. 2. The dendrimer shows an absorption band in the UV region around 270–280 nm and the free RB shows a peak at 552 nm (Fig. 2) (Fini et al. 2007). A weak band appearing around 500 nm is a measure of aggregation of the RB molecules in solution (Xu and Neckers 1989). On mixing equimolar amounts of dendrimer and the drug, the band appearing in the visible region (around 552 nm) is shifted towards 545 nm. Because of the movement of drug molecules into the dendritic entity, noncovalent interactions between the RB and the internal cavities of PAMAM dendrimers make such a blue shift (Cheng et al. 2007). In addition to this, intermolecular interaction between the carboxyl group of RB and the terminal groups of PAMAM dendrimers also makes such blue shift (Gigimol and Mathew 2007). In the UV–vis spectra of the G2.5 PAMAM + RB, there is no observation of broad shoulder peak around 500 nm since on encapsulation, the delocalization of the lone pair of electrons present in the RB molecule with dendrimer (Finia et al. 2004). These results concluded that the RB molecules interact well with G2.5 PAMAM dendrimers through cavity encapsulation.
FTIR spectroscopy is also used to study the host–guest interactions. The FTIR spectra of G2.5 PAMAM dendrimer, free RB and the G2.5 PAMAM + RB are shown in Fig. 3. The FTIR of G2.5 PAMAM dendrimer shows characteristic C = O stretching vibrations around 1,729 cm−1 that is assigned to an ester group (present as free terminal group in G2.5 dendrimer). The band at 1,641 cm−1 is assigned to N–H deformation vibration present in the amide group (Kolhe et al. 2003). In addition to this, a 1,000 cm−1 to 1,200 cm−1 band is assigned to the C–O stretching mode. Peaks in the region 2,800–3,200 cm−1 corresponds to N–H stretching and C–H stretching vibrations (Devarakonda et al. 2007). The FTIR spectra of RB shows a characteristic C = O stretching at 1,620 cm−1 (Jhonsi et al. 2009). All the bands were found individually on the dendrimer and the drug shifts if both are mixed together. The C = O and N–H deformation bands shifted to lower frequencies 1,718 cm−1 and 1,540 cm−1 that could be explained by intermolecular hydrogen bonding between electronegative atoms in the RB with the G2.5 PAMAM dendrimer. These observations reveal that the RB molecules are encapsulated in the dendritic box through their carboxyl group via electrostatic interaction.
Further, the interaction between RB and PAMAM dendrimer was also studied by fluorescence quenching measurements. Figure 4 represents the effect of PAMAM dendrimer on the fluorescence spectra of RB. The G2.5 PAMAM + RB showed decreased emission intensity, compared to free RB. Further, there is no band shift in the spectra of dendrimer–RB complex compared to free RB, indicating that no structural change occurred. The quenching observed is due to the electronegative carboxyl group of RB, which increased the interaction of dye with the dendrimer (Jhonsi et al. 2009). Zeta potential measurements enable the understanding of the interaction of RB and dendrimer moieties. G2.5 PAMAM dendrimer possesses a surface charge of +0.168 mV. Upon interaction with RB the surface charge of the dendrimer changed to −1.33 mV. This may be due to the presence of surface attached RB molecules that are having more electronegative chlorine atoms and a keto group in the molecule. Hence, the above characterization techniques reveal that the RB molecules are encapsulated in the dendritic cavity and also some of the RB molecules are absorbed at the terminal surface of the PAMAM dendrimers.
3.3 Drug loading and encapsulation efficiency of G2.5 PAMAM + RB nanocapsules
High drug loading and better encapsulation efficiency is expected for an ideal drug delivery agent, thereby, reducing the quantity of the matrix materials for drug administration (Mohanraj and Chen 2006). The drug loading in the G2.5 PAMAM + RB is through the absorption technique. Hence, the delivery system should be ideal in case of drug loading efficiency and drug release kinetics for carrying PDT, not suppressing the quantum yield of the PS after encapsulation. The amount of drug loaded into the dendrimer and the encapsulation efficiency of the G2.5 PAMAM dendrimers were measured spectrophotometrically during purification of G2.5 PAMAM + RB by centrifugation and are to be observed as 1.8% and 92.5% respectively.
3.4 In vitro drug release kinetics of G2.5 PAMAM + RB
In our G2.5 PAMAM + RB, the RB molecules are physically encapsulated in the cavity of the PAMAM dendrimer and a minimum quantity is absorbed at the terminal surface. The possible drug release kinetics is through diffusion process (Kedar et al. 2010). Figure 5 shows the in vitro drug release profile of the RB encapsulated G2.5 PAMAM dendrimers followed spectrophotometrically for a period of 72 h. It is observed that the systematic release of RB after 12, 24 and 48 h are 35%, 50% and 74% respectively. After 72 h, 83% of drug release is noticed. From the graph, it is clear that the rate of release of RB from the dendrimer at the initial stage is high whereas on the final stage it is found as low. The quicker release in the initial hours is the release of small amount of RB attached to the surface groups of the dendrimer. The drug release becomes somewhat slower, i.e., after 12 h is probably due to the encapsulated drug that is present in the dendritic cavity or the inner core of PAMAM. Sustained release was noticed after 48 h. The drug release studies shows that G2.5 PAMAM dendritic system possesses excellent controlled release properties suitable for carrying PDT of hydrophilic photosensitizers.
3.5 ROS quantum efficiency of G2.5 PAMAM + RB
The significant factor influencing the PDT efficiency is the quantum yield of ROS generation from the PS. The ROS generation of free RB and its encapsulated form was evaluated by determining the quantum yield by iodide method. It is experimentally found that the 1O2 quantum yield for free RB is 0.76. The 1O2 quantum yield for G2.5 PAMAM + RB was measured as 0.71 from using the iodide method (Mosinger and Micka 1997). Figure 6 depicts the graphical representation of the change in absorbance of the iodide band (351 nm) against the irradiation time for variously concentrated solutions of RB encapsulated in G2.5 PAMAM dendrimers. It is clear from Fig. 6. that the absorption of the iodide band increases with the increase in the concentration of the G2.5 PAMAM + RB, which indirectly show the increase in the ROS generation of G2.5 PAMAM + RB at higher concentration. These results strongly demonstrate that the G2.5 PAMAM can be an ideal drug delivery agent since no such alteration in the ROS activity is noticed when compared to the activity of the free RB.
3.6 Phototoxicity and dark toxicity of G2.5 PAMAM + RB
The aim was to investigate the photosensitizing activity of G2.5 PAMAM + RB against DLA cells and its efficiency as a potential PDT treatment for cancer. The cell viability of DLA cells upon photoirradiation as a function of concentration of G2.5 PAMAM + RB indicates the photodynamic effect in vitro. Figure 7 illustrates the photodynamic effect on the DLA cell line, as a function of the photosensitizer concentration. The free RB produced the lower photodynamic effect when compared to G2.5PAMAM + RB. The beneficial effect of the RB loaded dendrimers was mainly highlighted at a RB concentration of 510 nM. When the doses of the free RB and the G2.5 PAMAM + RB are increased to 510 nM, it was found that the cell viability percentage for G2.5 PAMAM + RB-treated cells was 24.9% when compared to the 38% cell viability for free RB-treated cells. The phototoxicity results show that the G2.5 PAMAM + RB are more toxic to the DLA cells compared to the toxicity of free RB. PAMAM dendrimer is a hyperbranched molecule and the amount of drug loading is as low as 1.8%, which ensures the uniform distribution of the photosensitizer in the dendritic matrix and this enables sustained drug release kinetics suitable for carrying PDT.
Low dark toxicity is one of the significant criteria for assessing the usefulness of photosensitizers, since the major side effects in clinical PDT result from the dark toxicity of photosensitizer to normal tissue. From Fig. 8, at lower concentration of the free RB, the cell viability is 92% and an increase in concentration of the free RB to 510 nM makes it more toxic and the cell viability is reduced to 56%. The dark toxicity of the RB-loaded dendritic nanostructures exhibits nontoxic at lower concentrations and is less toxic at higher concentrations (the cell viability is 69.8% at 510 nM) as compared to the dark toxicity of free RB. The very low toxicity of G2.5 PAMAM + RB might result from the good biocompatibility and low toxicity of PAMAM dendrimers (Koda et al. 2008; Bechet et al. 2008; Svenson and Tomalia 2005). It can be highlighted that differential toxicity was observed for G2.5 PAMAM + RB in the presence and absence of light against cancer cells. Therefore, the efficient photodynamic efficacy of G2.5 PAMAM + RB, together with the above-mentioned advantages, makes this type of formulation for delivery of photosensitizer in PDT potentially very useful for clinical application.