Mixed-Generation PAMAM G3-G0 Megamer as a Drug Delivery System for Nimesulide: Antitumor Activity of the Conjugate Against Human Squamous Carcinoma and Glioblastoma Cells

Polyhydroxylated dendrimer was synthesized from poly(amidoamine) (PAMAM) dendrimer generation 3 by addition of glycidol (G3gl). G3gl megamer was further modified by binding PAMAM G0 dendrimers by activation of G3gl with p-nitrophenylchloroformate, followed by the addition of excess PAMAM G0 and purification using dialysis. The maximum G0 binding capacity of G3gl was 12 in the case when G0 was equipped with two covalently attached nimesulide equivalents. Nimesulide (N) was converted into N-(p-nitrophenyl) carbonate derivative and fully characterized using X-ray crystallography and spectral methods. Nimesulide was then attached to G0 via a urea bond to yield G02N. The mixed generation G3gl–G02N megamer was characterized using 1H NMR spectroscopy, and its molecular weight was estimated to be 22.4 kDa. The AFM image of G3gl–G02N deposited on mica demonstrated aggregation of nimesulide-covered megamer. The height of the deposited megamer was 8.5 nm. The megameric conjugate with nimesulide was tested in vitro on three human cell lines: squamous cell carcinoma (SCC-15) and glioblastoma (U-118 MG) overexpressing cyclooxygenase-2 (COX-2), and normal skin fibroblasts (BJ). The conjugate efficiently penetrated into all cells and was more cytotoxic against SCC-15 than against BJ. Moreover, the conjugate produced a strong and selective antiproliferative effect on both cancer cell lines (IC50 < 7.5 µM).


Introduction
Nonsteroidal anti-inflammatory drugs (NSAID) are used for treatment of pain and inflammation [1]. Some of them, like celecoxib (C) or nimesulid (N) are selective or preferential cyclooxygenase-2 (COX-2) inhibitors. Over-expression of COX-2 has been detected in a variety of human tumors in breast, prostate, lung, skin and colon, and therefore, C and N are currently being tested for anticancer activity [2].
Side effects that can occur during long-term oral administration of NSAID include gastrointestinal irritation, ulcers, and impairment of renal blood circulation and glomerulic filtration. These adverse effects can be avoided by blood administration of C and N attached to a macromolecular carrier. We have followed the enhanced permeation and retardation (EPR) strategy and recently tested C and F-Moc-L-Leucine (L), the PPAR-γ agonist attached to third generation polyamidoamine dendrimer (PAMAM G3), in vitro for cytotoxicity against the cancer lines glioblastoma (U-118 MG) and squamous cell carcinoma . We found that the biotinylated conjugate bearing both C and L showed additive cytotoxicity for fibroblasts and both cancer lines in order BJ > U-118 MG > SCC- 15,with IC 50 in the range 0.69, 1.44 and 2.22 µM, respectively [3]. In addition, we also applied biotinylated PAMAM G3 as carrier of N [4]. The bioconjugates G3 N bearing 18 or 31 N exerted selective toxic effect on SCC-15 cells but not against normal human fibroblasts (BJ) at low range of concentration (1.25-10 µM) with 250 fold stronger action than native nimesulide. Moreover, this conjugates induced apoptotic cell death via COX-2/PGE 2 independent pathway.
On the other hand, amine groups of full generation PAMAM dendrimers are convenient sites for linking drug molecules or combinations of drugs, targeting molecules like biotin, folate and others.
PAMAM dendrimers can be stored in methanol at low temperatures, nonetheless, after several months, spontaneous aggregation of pristine dendrimers is observed [7]. The aggregation can be performed efficiently under chemical control using an appropriate linker, like glutaraldehyde [8,9], cyclooctyne-azide click coupling [10], or a straightforward reaction between variably terminated dendrimers, like G2-COOH and G2-NH 2 [7,11]. The stoichiometry and aggregation level in these procedures cannot be strictly controlled; therefore, high molecular weight dispersion of megamers is an inevitable feature.
We began our contribution to the field by using regular PAMAM G3 dendrimer as carrier of retinal, biotin and pyridoxal for transdermal delivery [12,13] and later applied them as carriers for anticancer drugs [3]; glucoheptoamidated PAMAM G3 dendrimer was shown to be an effective carrier for doxorubicin [6,14].
Encouraged by the results on the selective and efficient anticancer activity of PAMAM G3 N conjugates, we synthesized a new macromolecular carrier for N, comprised of hydroxylated PAMAM G3 with covalently bound PAMAM G0 dendrimers substituted with N. In this way, we obtained a very well water soluble megameric conjugate and tested it for cytotoxicity against human squamous carcinoma (SCC-15) and glioblastoma cells (U-118 MG), and comparatively against normal human fibroblasts (BJ).

Reaction of PAMAM Dendrimers with Glycidol
2,3-Epoxypropanol (glycidol, gl) is an oxirane which undergoes facile reaction with amine groups. The addition leads to gl epoxide ring opening and doubles the number of free hydroxyl groups. Therefore, gl found application in obtaining water-soluble oligoetherols from 1,3,5-triazine or barbituric acid, which were further used to obtain polyurethane foams with improved thermal resistance [15].
We have used gl to convert PAMAM G3 dendrimer into a completely hydroxyalkylated derivative, which remained water soluble like starting G3 and has no free primary amine groups. In order to establish the protocol for this conversion, we present a synthetic path starting from PAMAM G0. We found that eight molecules of gl are added within two days of reaction with G0 at room temperature to give the yellow oily product G0 gl (Scheme 1). The progress of reaction was monitored using 1 H NMR spectroscopy. The spectra of substrates gl and G0 are presented at Figure 1 (traces A and B, respectively). Large magnetic non-equivalence of both a and c methylene groups vicinal to chiral carbon b in gl (trace A) is almost cancelled after ring opening and addition of gl to G0. The largest chemical shift (0.5 ppm downfield) occurs for the H b proton upon conversion from gl into 1,2-dihydroxypropyl dihydroxypropyl group in G0 gl . The H7 resonance of G0 resonances is also shifted 0.1 ppm downfield upon addition of two gl onto the terminal nitrogen atom of G0 gl (trace C versus B).
The same protocol was used to convert G3 into G3 gl (Scheme 2). The product was purified by extensive dialysis against water and characterized using 1 H NMR spectroscopy. The spectrum of G3 gl is shown in Figure 1, trace D. The spectrum of G3 gl is similar to that of G0 gl except for the intensities of resonances from the 2,3-dihydropropyl protons, which correspond to [64H]  upon addition of two gl onto the terminal nitrogen atom of G0 gl (trace C versus B). The same protocol was used to convert G3 into G3 gl (Scheme 2). The product was purified by extensive dialysis against water and characterized using 1 H NMR spectroscopy. The spectrum of G3 gl is shown in Figure 1, trace D. The spectrum of G3 gl is similar to that of G0 gl except for the intensities of resonances from the 2,3-dihydropropyl protons, which correspond to [64H] for b' and [128H] for a'and c' in comparison with PAMAM G3 -CH2-COresonance of intensity    Scheme 2. Conversion of PAMAM G3 dendrimer to G3 gl by hydroxyalkylation with glycidol. PAMAM G3 has 32 terminal amine groups (n = 32) that each react with two molecules of gl to give a total of 64 2,3-dihydroxypropyl substituents with 128 free hydroxyl groups.
The 1 H and 13 C NMR spectra of both G0 gl and G3 gl are quite complicated due to the racemic gl substrate used for synthesis. Therefore many stereoisomers are formed at every terminal disubstituted nitrogen, containing an attached chiral gl in combinations RR, SS, RS, and SR local chirality. When considering only four microstates, two spectra of gl substituents can be expectedone from two meso forms and a second from chiral RR and SS microstates. Because more than two spectra are observed, it is obvious that two arms of the outer sphere of dendrimer influence the symmetry of NMR spectra. Namely, 16 microstates must be considered: RRRR; RRRS; …RSSS; SSSS, from which six various spectra of relative intensity, 1:1:1:1:2:2, should be observed if no enantiodiscrimination of chiral gl addition occurs. Still, the 13 C NMR spectra show at least four sets of resonances. Additionally, their intensities do not follow the 1:1:1:1:2:2 ratio indicating that a stereochemical discrimination factor for a second gl addition influences the populations of microstates. We are currently working on determination of gl addition enantioselectivity. Despite the structural complexity of G3 gl , the chemical availability of the terminal hydroxyl group of the 2,3dihydroxypropyl substituents for further functionalization enables further expansion of the G3 gl dendrimer.

Nimesulide Activation and Attachment to PAMAM G0
p-Nitrophenyl chloroformate (NPCF) provides a one-carbon linker to amine groups of PAMAM dendrimers. It has been shown that the copolymer obtained from butylene oxide and ethylene oxide was efficiently activated with NPCF, followed by covalent attachment of PAMAM G1-G3 dendrimers [16].
Nimesulide was activated by obtaining the N-(4-nitrophenoxycarbonyl) derivative 1 from reaction of NPCF with N. Derivative 1 was fully characterized using NMR, mass spectrometry, and crystallography (Scheme 3). The schematic view of 1 is shown in Figure 2. Scheme 3. Reaction pathway of nimesulide derivatization with p-nitrophenyl chloroformate (NPCF) with atom numbering of 1.

Scheme 2.
Conversion of PAMAM G3 dendrimer to G3 gl by hydroxyalkylation with glycidol. PAMAM G3 has 32 terminal amine groups (n = 32) that each react with two molecules of gl to give a total of 64 2,3-dihydroxypropyl substituents with 128 free hydroxyl groups. from which six various spectra of relative intensity, 1:1:1:1:2:2, should be observed if no enantiodiscrimination of chiral gl addition occurs. Still, the 13 C NMR spectra show at least four sets of resonances. Additionally, their intensities do not follow the 1:1:1:1:2:2 ratio indicating that a stereochemical discrimination factor for a second gl addition influences the populations of microstates. We are currently working on determination of gl addition enantioselectivity. Despite the structural complexity of G3 gl , the chemical availability of the terminal hydroxyl group of the 2,3dihydroxypropyl substituents for further functionalization enables further expansion of the G3 gl dendrimer.

Nimesulide Activation and Attachment to PAMAM G0
p-Nitrophenyl chloroformate (NPCF) provides a one-carbon linker to amine groups of PAMAM dendrimers. It has been shown that the copolymer obtained from butylene oxide and ethylene oxide was efficiently activated with NPCF, followed by covalent attachment of PAMAM G1-G3 dendrimers [16].
Nimesulide was activated by obtaining the N-(4-nitrophenoxycarbonyl) derivative 1 from reaction of NPCF with N. Derivative 1 was fully characterized using NMR, mass spectrometry, and crystallography (Scheme 3). The schematic view of 1 is shown in Figure 2.  The same protocol was used to convert G3 into G3 gl (Scheme 2). The product was purified by extensive dialysis against water and characterized using 1 H NMR spectroscopy. The spectrum of G3 gl is shown in Figure 1, trace D. The spectrum of G3 gl is similar to that of G0 gl except for the intensities of resonances from the 2,3-dihydropropyl protons, which correspond to [64H] for b' and [128H] for a'and c' in comparison with PAMAM G3 -CH 2 -CO-resonance of intensity [120H] at 2.2 ppm (H-4').
The 1 H and 13 C NMR spectra of both G0 gl and G3 gl are quite complicated due to the racemic gl substrate used for synthesis. Therefore many stereoisomers are formed at every terminal di-substituted nitrogen, containing an attached chiral gl in combinations RR, SS, RS, and SR local chirality. When considering only four microstates, two spectra of gl substituents can be expected-one from two meso forms and a second from chiral RR and SS microstates. Because more than two spectra are observed, it is obvious that two arms of the outer sphere of dendrimer influence the symmetry of NMR spectra. Namely, 16 microstates must be considered: RRRR; RRRS; . . . RSSS; SSSS, from which six various spectra of relative intensity, 1:1:1:1:2:2, should be observed if no enantiodiscrimination of chiral gl addition occurs. Still, the 13 C NMR spectra show at least four sets of resonances. Additionally, their intensities do not follow the 1:1:1:1:2:2 ratio indicating that a stereochemical discrimination factor for a second gl addition influences the populations of microstates. We are currently working on determination of gl addition enantioselectivity. Despite the structural complexity of G3 gl , the chemical availability of the terminal hydroxyl group of the 2,3-dihydroxypropyl substituents for further functionalization enables further expansion of the G3 gl dendrimer.

Nimesulide Activation and Attachment to PAMAM G0
p-Nitrophenyl chloroformate (NPCF) provides a one-carbon linker to amine groups of PAMAM dendrimers. It has been shown that the copolymer obtained from butylene oxide and ethylene oxide was efficiently activated with NPCF, followed by covalent attachment of PAMAM G1-G3 dendrimers [16].
Nimesulide was activated by obtaining the N-(4-nitrophenoxycarbonyl) derivative 1 from reaction of NPCF with N. Derivative 1 was fully characterized using NMR, mass spectrometry, and crystallography (Scheme 3). The schematic view of 1 is shown in Figure 2.  Table A1 (Appendix A).
The X-ray structure of N was reported in 1995 [17]. Significant changes in bond lengths occur around the central sulfonamide nitrogen upon replacement of the hydrogen atom by 4nitrophenylcarbonate in 1, namely elongation of the N-S bond from 1.64 to 1.69 Å, N-C2 bond from 1.41 to 1.45 Å, and S-C1 from 1.47 to 1.75 Å. The torsion angle S-N-C2 tightens from 124.7° in N to 118.3° in 1 due to steric hindrance of the 4-nitrophenylcarbonate substituent. We have noticed that 1 is stable in aqueous solution but reacts readily with amine groups on PAMAM G0.
Thus, 1 was used to covalently attach N via one-atom linker to PAMAM G0 to give G0 substituted with two molecules of N (Scheme 4). The urea bond formation was accompanied by release of 4-nitrophenol, which was removed by rinsing with chloroform. The characteristic IR carbamate ν(CO) band centered at 1755 cm -1 in 1 disappeared in G0 2N and was replaced by a ν(CO) band centered at 1644 cm −1 from G0 amide and a urea bond between N and G0 ( Figure A1). Obtained G0 2N was further used to attach it to core G3 gl after its activation with 14 equivalents of NPCF.    Table A1 (Appendix A).
The X-ray structure of N was reported in 1995 [17]. Significant changes in bond lengths occur around the central sulfonamide nitrogen upon replacement of the hydrogen atom by 4-nitrophenylcarbonate in 1, namely elongation of the N-S bond from 1.64 to 1.69 Å, N-C 2 bond from 1.41 to 1.45 Å, and S-C 1 from 1.47 to 1.75 Å. The torsion angle S-N-C 2 tightens from 124.7 • in N to 118.3 • in 1 due to steric hindrance of the 4-nitrophenylcarbonate substituent. We have noticed that 1 is stable in aqueous solution but reacts readily with amine groups on PAMAM G0.
Thus, 1 was used to covalently attach N via one-atom linker to PAMAM G0 to give G0 substituted with two molecules of N (Scheme 4). The urea bond formation was accompanied by release of 4-nitrophenol, which was removed by rinsing with chloroform. The characteristic IR carbamate ν(CO) band centered at 1755 cm −1 in 1 disappeared in G0 2N and was replaced by a ν(CO) band centered at 1644 cm −1 from G0 amide and a urea bond between N and G0 ( Figure A1). Obtained G0 2N was further used to attach it to core G3 gl after its activation with 14 equivalents of NPCF.  Table A1 (Appendix A).
The X-ray structure of N was reported in 1995 [17]. Significant changes in bond lengths occur around the central sulfonamide nitrogen upon replacement of the hydrogen atom by 4nitrophenylcarbonate in 1, namely elongation of the N-S bond from 1.64 to 1.69 Å, N-C2 bond from 1.41 to 1.45 Å, and S-C1 from 1.47 to 1.75 Å. The torsion angle S-N-C2 tightens from 124.7° in N to 118.3° in 1 due to steric hindrance of the 4-nitrophenylcarbonate substituent. We have noticed that 1 is stable in aqueous solution but reacts readily with amine groups on PAMAM G0.
Thus, 1 was used to covalently attach N via one-atom linker to PAMAM G0 to give G0 substituted with two molecules of N (Scheme 4). The urea bond formation was accompanied by release of 4-nitrophenol, which was removed by rinsing with chloroform. The characteristic IR carbamate ν(CO) band centered at 1755 cm -1 in 1 disappeared in G0 2N and was replaced by a ν(CO) band centered at 1644 cm −1 from G0 amide and a urea bond between N and G0 ( Figure A1). Obtained G0 2N was further used to attach it to core G3 gl after its activation with 14 equivalents of NPCF.

Synthesis of Megamer with Conjugated G0
G3 gl was activated with 14 equivalents of NPCF in dry dimethylsulfoxide in the presence of excess TEA and used without isolation to bind 12 equivalents of G0 2N (Scheme 5). G3 gl was activated with 14 equivalents of NPCF in dry dimethylsulfoxide in the presence of excess TEA and used without isolation to bind 12 equivalents of G0 2N (Scheme 5 Scheme 5. Synthesis scheme of megamer composed of G3 gl core (n = 32), to which one-carbon linker is provided by reaction with p-nitrophenyl chloroformate (14 equivalents, m = 12). Here, NMS represents nimesulide.
The mixture was dialyzed extensively against water. The stoichiometry of the obtained megameric conjugate was determined by the 1 H NMR spectrum of the product ( Figure 3). Total integral intensity of CH resonances of G3 gl corresponds to [804H] while intensities of OH and NH signals are [128H] and ca [60H], respectively (trace A). In the 1 H NMR spectrum of G3 G02N , the nimesulide aromatic proton resonance intensity (in the 8-7 ppm region) corresponds to [192H] (24 N attached) versus broad CH signals intensity [1308H] in the 4-2 ppm region (trace B). The core G3 64gl contribution is [804H], while additional intensity derives from 12 G0 ([432H]) and methyl groups of N ([72H]). The OH proton resonance is spread over the 4.25-5.25 ppm region and therefore integration of it is unreliable, as are NH broad resonances centered at 7.84 ppm. Thus, the average megameric conjugate bears 24 N prodrug units linked to G0 via urea bonds, while G0 2N subunits are linked to the G3 gl core by carbamate bonds. IR spectra of the G3 gl core show a strong carbonyl stretching vibration band ν(CO) centered at 1634 cm −1 , while ν(CO) for the urea bond (maximum at 1644 cm -1 ) in G0 2N is overlapped with that of amide ν(CO) of G0. The ν(CO) of the carbamate bond between G3 gl and G0 in megamers was observed at 1733 cm −1 , well separated from other ν(CO) bands ( Figure A1). Scheme 5. Synthesis scheme of megamer composed of G3 gl core (n = 32), to which one-carbon linker is provided by reaction with p-nitrophenyl chloroformate (14 equivalents, m = 12). Here, NMS represents nimesulide.
The mixture was dialyzed extensively against water. The stoichiometry of the obtained megameric conjugate was determined by the 1 H NMR spectrum of the product ( Figure 3 ). The OH proton resonance is spread over the 4.25-5.25 ppm region and therefore integration of it is unreliable, as are NH broad resonances centered at 7.84 ppm. Thus, the average megameric conjugate bears 24 N prodrug units linked to G0 via urea bonds, while G0 2N subunits are linked to the G3 gl core by carbamate bonds. IR spectra of the G3 gl core show a strong carbonyl stretching vibration band ν(CO) centered at 1634 cm −1 , while ν(CO) for the urea bond (maximum at 1644 cm −1 ) in G0 2N is overlapped with that of amide ν(CO) of G0. The ν(CO) of the carbamate bond between G3 gl and G0 in megamers was observed at 1733 cm −1 , well separated from other ν(CO) bands ( Figure A1). A similar synthesis of megameric conjugate was performed for FITC-labeled G0 (G0 F ). We deliberately used only four equivalents of NPCF to activate G0 gl and 4 equivalents of G0 F to obtain G3 G0F for preliminary confocal microscopy studies on cell cultures.

Molecular Weight and Size of Dendrimers and Megamers
The dispersity of molecular weight, molecular shape and size are important factors influencing the ability of the drug carrier to cross cell membranes by endocytosis. We determined the molecular weight of the obtained megamers, including the series of G2 gl -G0, and G3 gl -G0 synthesized in order to optimize the reagent stoichiometry in two-step synthesis followed by long purification of megamers by dialysis with water. The molecular weight and dispersity of megamers was determined using gel permeation chromatography (GPC) with dimethylformamide eluent. The column was calibrated with a series of G2-G5 dendrimers substituted with glycidol, for which we determined the average composition using 1 H NMR spectra. Thus the reference compounds were very similar chemically to the dendrimers tested.
We have found that G3 gh , upon reaction with 18 equivalents of NPCF followed by addition of 20 equivalents of G0 to give G3 G0 , showed Mw corresponding to stoichiometry 1:13, while after prolonged dialysis of the same sample, the stoichiometry dropped to 1:10 (samples are named G3 G0 b before and G3 G0 a after dialysis, see Figure A2 and Table A2). Also using GPC, we found that G3:G0 stoichiometry in the G3 G02N megamer was 1:9, which corresponds to 18 N equivalents per one megamer. This is very similar stoichiometry to that obtained from integration of resonances of N in A similar synthesis of megameric conjugate was performed for FITC-labeled G0 (G0 F ). We deliberately used only four equivalents of NPCF to activate G0 gl and 4 equivalents of G0 F to obtain G3 G0F for preliminary confocal microscopy studies on cell cultures.

Molecular Weight and Size of Dendrimers and Megamers
The dispersity of molecular weight, molecular shape and size are important factors influencing the ability of the drug carrier to cross cell membranes by endocytosis. We determined the molecular weight of the obtained megamers, including the series of G2 gl -G0, and G3 gl -G0 synthesized in order to optimize the reagent stoichiometry in two-step synthesis followed by long purification of megamers by dialysis with water. The molecular weight and dispersity of megamers was determined using gel permeation chromatography (GPC) with dimethylformamide eluent. The column was calibrated with a series of G2-G5 dendrimers substituted with glycidol, for which we determined the average composition using 1 H NMR spectra. Thus the reference compounds were very similar chemically to the dendrimers tested.
We have found that G3 gh , upon reaction with 18 equivalents of NPCF followed by addition of 20 equivalents of G0 to give G3 G0 , showed M w corresponding to stoichiometry 1:13, while after prolonged dialysis of the same sample, the stoichiometry dropped to 1:10 (samples are named G3 G0 b before and G3 G0 a after dialysis, see Figure A2 and Table A2). Also using GPC, we found that G3:G0 stoichiometry in the G3 G02N megamer was 1:9, which corresponds to 18 N equivalents per one megamer. This is very similar stoichiometry to that obtained from integration of resonances of N in 1 H NMR spectrum of G3 G02N megamer (vide supra). Thus the prolonged dialytic purification of megamers with water resulted in ca 10-25% loss of G0 from a megamer.
The molecular size of the megamers was estimated using the DLS method and compared to a series of GM gl dendrimers synthesized to serve as a reference series, in addition to known GM dendrimers (where M is generation 2-5). The DLS-estimated diameters for dendrimers and megamer (determined in volume mode) are collected in Table A3. The G3 G02N megamer diameter was 5.07 ± 0.14 nm, which is similar to that of G4 gl (5.12 nm).
A linear relationship was found by plotting the measured height of dendrimers as a function of molecular weight with high correlation (R 2 = 0.9993) ( Figure 4F). To determine the distribution of acquired numerical data, histograms were plotted and a Jarque-Bera test was performed. None of data series describing vertical distance of dendrimers comes from a normal distribution (p-value < 0.05). The set of vertical distance data was also tested for the presence of outliers by means of a modified Thompson tau test. For all dendrimers, data outliers were found at the 95% confidence level. Occurrence of outliers may be evidence of gradual aggregation of dendrimers, which take the form of larger objects on the mica surface.

Cytotoxicity
To evaluate the utility of synthesized megameric conjugate G3 G0N containing the cyclooxygenase-2 (COX-2) inhibitor nimesulide, three human cell lines were chosen: squamous carcinoma (SCC-15) and glioblastoma (U-118 MG), both overexpressing COX-2, and comparative normal human skin fibroblasts (BJ) with lower expression of COX-2 [3]. The cytotoxicity assay revealed inhibitory action of the studied conjugate at low 7.5 µM concentration and, in the case of squamous carcinoma cells (SCC-15), even as low as 3.75 µM, after 24 h of exposure. At the highest concentration (15 µM) all cell lines revealed about 50% decrease in viability. The most resistant were glioma cells and the least SCC-15 cells ( Figure 5). Over the entire concentration range, the viability of squamous cell carcinomas was noticeably lower than other cell lines, particularly glioma cells. Statistical analysis revealed, however, that significant differences appeared at concentrations of 3.75 and 7.5 µM. Viability of normal fibroblasts had an intermediate degree. Microscope images collected during the assay performance confirmed obtained results. Changes in cell morphology were seen as concentration dependent cell shrinkage and decrease in cell number, cellular protrusion size and neutral red content in the lysosomes. The described symptoms of cell degradation were the most visible in SCC-15 cells. The biological effect of G3 G02N appeared probably from the presence of N on the G3 G02N surface. As described in many papers, N, a preferable COX-2 inhibitor, is a promising chemopreventive and antineoplastic agent that acts via blocking COX-2 and decreasing the concentration of prostaglandins inside the tumor or via other COX-independent pathways [18,19]. It was assumed that SCC-15 and U-118 MG cells with elevated COX-2 levels [3] would be more sensitive to a megamer containing N. SCC-15 cells showed a significant stronger decrease of viability at 3.75 µM than normal cells with a lower COX-2 level. However, glioma cells did not react more strongly than BJ cells. Slightly more light is shed on this problem by our recent studies, where we showed that the action of nimesulide-substituted PAMAM G3 dendrimers selectively reduced the viability of SCC-15 cells compared to normal fibroblasts. However, this effect was not based on the COX-2 and PGE 2 axis [4]. Therefore, it should be assumed that the action of the nimesulide substituted PAMAM dendrimers, including the tested megamer, can be also realized via independent COX-2 and PGE 2 pathways and requires further, profound studies of this issue. Moreover, both PAMAM G0 and G3 dendrimers showed much lower toxicity to the cancer cells compared to obtained G3 G02N . G0 PAMAM dendrimers were not toxic against colorectal adenocarcinoma (Caco-2) cells up to 10 mM concentration after 3 h of incubation [20]. In addition, Zeng et al. demonstrated lack of G0 PAMAM dendrimer toxicity in human neural progenitor cells (hNPCs) up to 200 µM concentration after 24 or even 72 h of incubation [21]. The PAMAM G3 IC50 for normal human fibroblasts (BJ) after 24 h incubation was higher than for SCC-15 cells (7.5 and 30 µM, respectively) [22]. Attachment of the PAMAM G0 dendrimer molecules with N to the G3 gl dendrimer resulted in a reversal of the biological effect on squamous carcinoma cells, which became more sensitive than normal human fibroblasts. Moreover, both PAMAM G0 and G3 dendrimers showed much lower toxicity to the cancer cells compared to obtained G3 G02N . G0 PAMAM dendrimers were not toxic against colorectal adenocarcinoma (Caco-2) cells up to 10 mM concentration after 3 h of incubation [20]. In addition, Zeng et al. demonstrated lack of G0 PAMAM dendrimer toxicity in human neural progenitor cells (hNPCs) up to 200 µM concentration after 24 or even 72 h of incubation [21]. The PAMAM G3 IC 50 for normal human fibroblasts (BJ) after 24 h incubation was higher than for SCC-15 cells (7.5 and 30 µM, respectively) [22]. Attachment of the PAMAM G0 dendrimer molecules with N to the G3 gl dendrimer resulted in a reversal of the biological effect on squamous carcinoma cells, which became more sensitive than normal human fibroblasts.

Cellular Accumulation
Fluorescent labeled megamer (G3 G02N *) penetrated into all three cell lines efficiently, but the profile of its cellular accumulation differed in particular cell types. The most efficient penetration of G3 G02N * was observed in SCC-15 cells at a concentration of 3.75 µM, similar to that in BJ and somewhat lower than in U-118 MG glioma cells ( Figure 6A,B).

Cellular Accumulation
Fluorescent labeled megamer (G3 G02N *) penetrated into all three cell lines efficiently, but the profile of its cellular accumulation differed in particular cell types. The most efficient penetration of G3 G02N * was observed in SCC-15 cells at a concentration of 3.75 µM, similar to that in BJ and somewhat lower than in U-118 MG glioma cells (Figures 6A and 6B). At 7.5 and 15 µM concentrations, the most efficient uptake was found in normal fibroblast cells, followed by SCC-15 and U-118 MG cells. The reason for this trend could be that BJ cells are more resistant to the toxic effects of G3 G02N and maintained a high degree of intracellular nanoparticle transport. Furthermore, phagocytic activity of these cells may be contributing to this phenomenon [23]. The lower accumulation of G3 G02N * in glioblastoma and SCC-15 cells may be caused by active xenobiotic efflux systems present in cancer cells, including gliomas [24]. The degree of fluorescently labelled megamer accumulation in mitochondria, nuclei or in other subcellular organelles after 24 h incubation were estimated with confocal microscopy by determining the degree of co-localization with fluorescently stained organelles. Studies revealed that G3 G02N* was present in intracellular compartments of all studied cell lines ( Figure 6C). Most of the megamer absorbed by the cells remained in endocytic vesicles and in lysosomal vesicles. A significant part of the dendrimer was also dispersed in the cytoplasm of the cells, especially at higher concentrations of G3 G02N* . Only a small part accumulated in the mitochondria in a concentration dependent manner. In At 7.5 and 15 µM concentrations, the most efficient uptake was found in normal fibroblast cells, followed by SCC-15 and U-118 MG cells. The reason for this trend could be that BJ cells are more resistant to the toxic effects of G3 G02N and maintained a high degree of intracellular nanoparticle transport. Furthermore, phagocytic activity of these cells may be contributing to this phenomenon [23]. The lower accumulation of G3 G02N * in glioblastoma and SCC-15 cells may be caused by active xenobiotic efflux systems present in cancer cells, including gliomas [24].
The degree of fluorescently labelled megamer accumulation in mitochondria, nuclei or in other subcellular organelles after 24 h incubation were estimated with confocal microscopy by determining the degree of co-localization with fluorescently stained organelles. Studies revealed that G3 G02N* was present in intracellular compartments of all studied cell lines ( Figure 6C). Most of the megamer absorbed by the cells remained in endocytic vesicles and in lysosomal vesicles. A significant part of the dendrimer was also dispersed in the cytoplasm of the cells, especially at higher concentrations of G3 G02N* . Only a small part accumulated in the mitochondria in a concentration dependent manner. In addition, G3 G02N* penetrated the mitochondria of normal fibroblasts and glioma cells to a larger degree than squamous carcinoma cells at all concentrations ( Figure 6C, yellow signal). The penetration of the megamer into the nuclei was negligible and only noticeable in the case of normal fibroblast cells at the highest concentration of 15 µM ( Figure 6C, light blue signal). Our findings are in agreement with the observations of others concerning PAMAM dendrimers [25,26]. These results confirm that the studied megamer, in addition to its anticancer properties, can be a proper agent to deliver drugs into resistant cancer cells.

Anti-Proliferation
In this study, the megamer substituted with nimesulide G3 G02N was synthesized as a highly promising anti-cancer agent due to its anti-proliferative properties. N exert a strong anti-proliferative effect by promoting cell cycle arrest in multiple gastric cancer cell lines [27]. Moreover, the anti-proliferative action of this highly preferable COX-2 inhibitor was demonstrated against non-small cell lung cancer, hepatoma SMMC-7721 cells, human gastric adenocarcinoma SGC7901 cells, human pancreatic cancer cells and human esophageal adenocarcinoma OE33 cells [18,28].
In this study, G3 G02N revealed strongly selective, antiproliferative action against cancer cells compared to normal fibroblasts after 72 h incubation (Figure 7). Mol. Sci. 2019, 20, 4998 13 of 24 addition, G3 G02N* penetrated the mitochondria of normal fibroblasts and glioma cells to a larger degree than squamous carcinoma cells at all concentrations ( Figure 6C, yellow signal). The penetration of the megamer into the nuclei was negligible and only noticeable in the case of normal fibroblast cells at the highest concentration of 15 µM ( Figure 6C, light blue signal). Our findings are in agreement with the observations of others concerning PAMAM dendrimers [25,26]. These results confirm that the studied megamer, in addition to its anticancer properties, can be a proper agent to deliver drugs into resistant cancer cells.

Anti-Proliferation
In this study, the megamer substituted with nimesulide G3 G02N was synthesized as a highly promising anti-cancer agent due to its anti-proliferative properties. N exert a strong anti-proliferative effect by promoting cell cycle arrest in multiple gastric cancer cell lines [27]. Moreover, the antiproliferative action of this highly preferable COX-2 inhibitor was demonstrated against non-small cell lung cancer, hepatoma SMMC-7721 cells, human gastric adenocarcinoma SGC7901 cells, human pancreatic cancer cells and human esophageal adenocarcinoma OE33 cells [18,28].
In this study, G3 G02N revealed strongly selective, antiproliferative action against cancer cells compared to normal fibroblasts after 72 h incubation (Figure 7).
The inhibition of cell division was particularly evident in the SCC-15 line and only slightly weaker in the case of glioblastoma cells, with IC50 values of 3.19 and 4.48 µM, respectively, compared to 9.28 µM for normal human fibroblasts. Likewise, the strong inhibitory action of nimesulide after 72 h incubation against human squamous carcinoma cells (A431) was observed by Khodaie et al. with IC50 = 250 µM [29]. Differences in IC50 values for native drug and G3 G02N suggest that the decrease of cell proliferation is affected not only by nimesulide itself but also additionally by its megameric carrier. It has been noticed that proliferation of normal BJ cells grows to 134% at 1.88 µM concentration. Similar cell growth enhancement was observed for PAMAM G2, G4, and G6 neat dendrimers in HeLa and HEK293T lines at 100-500 nM concentration of dendrimer [30].   [29]. Differences in IC 50 values for native drug and G3 G02N suggest that the decrease of cell proliferation is affected not only by nimesulide itself but also additionally by its megameric carrier. It has been noticed that proliferation of normal BJ cells grows to 134% at 1.88 µM concentration. Similar cell growth enhancement was observed for PAMAM G2, G4, and G6 neat dendrimers in HeLa and HEK293T lines at 100-500 nM concentration of dendrimer [30].

Synthesis of PAMAM G0 Dendrimer Substituted with 8 Glycidol Molecules, G0 gl
Glycidol (gl, 0.9 mL, 0.967 g, 13.0 mmol) was added dropwise to a solution of PAMAM G0 (0.884 g, 1.63 mmol) in 20 mL of methanol with magnetic stirring. The mixture was left at room temperature for 2 days. Then methanol and excess gl were removed by vacuum rotary evaporation. Yellow syrup was obtained, which was identified using 1 H NMR as G0 gl (Figure 1) Dendrimer PAMAM G3 in methanol (525 mg, 0.076 mmol in 5 mL; 15.2 mM solution) was added dropwise into a 25 mL round bottom flask containing glycidol (0.35 mL, 374 mg, 5.10 mmol) in 2 mL of methanol. The mixture was left at room temperature for 2 days. After two days, the solution was transferred into a nitrocellulose dialytic bag (MW cutoff = 3 kDa) and dialyzed against water for three days. Water was removed under reduced pressure by rotary evaporation and the resulting solid residue was dried under 0.2 mm Hg pressure overnight. A 90% yield (810 mg) of G3gl was obtained as a brown syrup and was characterized using 1 H NMR spectroscopy. The isolated G3 gl macromolecular core was found to be very soluble in water and in dimethyl sulfoxide. Nimesulide (220 mg, 0.713 mmole) was dissolved in chloroform (5 mL) and triethylamine (TEA, 0.4 mL). Then 4-nitrophenylchloroformate was added (288 mg, 1.43 mmoles) in portions with continuous stirring and the mixture was left under nitrogen at room temperature for 24 hrs. The chloroform layer was washed three times with water (25 mL) and the solvent was removed under reduced pressure. The mixture was chromatographed with chloroform: ethyl acetate (7:1) on silica gel. 1 was eluted as first fraction as identified using NMR and MS. Crystals of 1 were grown in an NMR tube upon layering the solution (0.05 M) with hexane. The percent yield of 1 was 52.9% (250 mg).
Analytical data (for atom numbering see 3.2.5. Synthesis of megamers G3 gl -12G0 2N and G3 gl -4G0 F G3 G02N : 100 mg G3 gl (8.5 µmoles) was dissolved in 2 mL of DMSO and 0.5 mL of TEA. To this solution, solid NPCF (28.2 mg, 102 µmoles) was added with vigorous stirring. The obtained mixture was immediately used to react with 119 mg (100 µmoles) of G0 2N in 2 mL DMSO at room temperature overnight. Then the mixture was dialyzed in nitrocellulose tubing (MW cutoff = 3 kDa) against water for 4 days. Water was removed under reduced pressure and the 1 H NMR spectrum was recorded. Based upon integral intensity of N versus PAMAM resonances the stoichiometry of conjugate was determined to be G3 gl -12G0 2N (see Figure 2 The G3 G02N conjugate was tested for toxicity against cancer cells. Additionally, ca 1 µmol of G3 G02N (26 mg) was labeled with one equivalent of FITC and used for confocal microscopic monitoring in cell lines (G3 G02N *, vide infra).
G3 G0F : 31 mg G3 gl (2.7 µmoles) was dissolved in 1.5 mL of DMSO and 0.3 mL of TEA. To this solution, solid NPCF (2.3 mg, 11.4 µmoles) was added with vigorous stirring. Then, 0.115 mL of 0.10 M G0 F solution in DMSO (11.5 µmoles) was added and the mixture left for 24 h at room temperature. Then the mixture was dialyzed against water for 3 days in the dark. The solvents were removed under reduced pressure, yielding 35 mg of G3 gl . G3 gl was conjugated with 4 equivalents of G0 F based upon integral intensity of F aromatic resonances versus total intensity of G3 and G0 resonances. The estimated yield was 84%. The average molecular weight determined using GPC was ca 15.5 kDa vs theoretical molecular weight 15,383 for G3-4G0 F stoichiometry. The aqueous solution of G3 G0F was used to deposit the monomolecular layer in mica to estimate molecular size using AFM (vide infra

Cell Cultures
Human squamous cell carcinoma (SCC-15) and human glioblastoma (U-118 MG) cell lines obtained from ATCC (Manassas, VA, USA) were cultured in DMEM (doubling time 48 and 35 h, respectively). Normal fibroblast (BJ) purchased from ATCC (doubling time 1.9 days) were grown in EMEM. Each medium was supplemented with 10% heat-inactivated FBS and 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were cultured as described [22]. All biological tests were carried out in triplicates in three independent experiments. in the corresponding culture media and added to cells (100 µL/well). The DMSO concentration was adjusted to 0.4% in all samples, which had no significant effect on treated cell lines. Following a 24 h incubation, an NR assay was performed as described before [32].

Cellular Accumulation of Megamer
Cells were seeded in 96-well plates at a density of 2 × 10 4 cells/well (BJ and U-118 MG) or 4 × 10 4 (SCC-15). After 24 h incubation cells were treated with G3 G02N * in the range of concentration 0-15 µM in complete medium for 24 h. After incubation plates were centrifuged (2000 rpm, 5 min), cells were washed with PBS and fixed with 3.7% formaldehyde. Then, 600 nM DAPI solution in PBS was added (100 µL/well) and incubated for 1 h at room temperature. Fluorescence signals were read at 485/530 nm for FITC and 360/460 nm for DAPI with an Infinite M200 PRO Microplate Reader (TECAN Group Ltd., Switzerland). The DAPI staining was used to estimate the number of cells and calculate fluorescence signals per equal cell number.

Confocal Microscopy
Cells were cultured on microscope chamber slides (Nunc, Denmark) for 48 h at a density of 60 × 10 4 or 1.2 × 10 5 cells (for BJ and U-118 MG or SCC-15 cells, respectively) in 400 µL of complete medium. FITC-labeled megamer was added at 0 to 15 µM concentrations (400 µL/well). After 24 h of incubation and washing (2×PBS), the cells were fixed with 3.7% formaldehyde for 15 min and stained with 600 nM DAPI solution in PBS (1 h, RT). Images from each well were collected using a confocal microscope (Olympus FV10i, Tokyo, Japan) at 488/530 nm for FITC, 405/461 nm for DAPI, and 644/665 nm for MitoTracker. Images were collected in the Z-axis position at the largest nuclear cross section area. Pinhole was set for 1 AU (airy unit) and the obtained images had an optical section thickness of approximately 1.02 µm. Image processing was performed with the ImageJ software (Bethesda, MD, USA).

Proliferation Assay
For assay, cells were seeded into 96-well microplates at a density of 5 × 10 3 cells/well and incubated for 24 h at 37 • C. After medium removal, G3 G02N solutions were prepared as described above in medium (200 µL/well). The plates were then incubated for 72 h. Then, plates were centrifuged (2000 rpm, 5 min). Cells were then washed with PBS and fixed in 3.7% formaldehyde solution in PBS and stained with 600 nM DAPI solution in PBS (100 µL/well, 1 h). The fluorescent signal, proportional to the number of cells, was measured in a Tecan Infinite M200 PRO Multimode Microplate Reader (TECAN Group Ltd., Switzerland) at 360/460 nm. The results were expressed as % of the control (DMSO treated cells).

Spectroscopy
1-D 1 H, 13 C NMR as well as 2-D 1 H-1 H COSY and 1 H-13 C HSQC and HMBC spectra were recorded with Bruker 300 MHz instrument. Gold enhanced target laser desorption ionization mass spectra were recorded with a Bruker Autoflex Speed reflectron time-of-flight mass spectrometer equipped with a SmartBeam II laser (352 nm) in 80-2080 m/z range [33].
IR spectra were taken with ALPHA FT-IR Bruker instrument in KBr pellets and ATR mode.

Atomic Force Microscopy Studies
AFM images were recorded in air with a Nanoscope IIId scanning probe microscope with Extender Module (Bruker). Standard tapping mode AFM probes (NanoAndMore, Watsonville, California, USA) were used with a resonance frequency in the range of 200-400 kHz, with a typical spring constant of 42 N/m and with a nominal apex radius of silicon tip curvature around 7 nm. The samples with dendrimers were placed on freshly cleaved ultra-clean mica (Nano and More) and incubated at room temperature for 60 sec. The mica discs were then rinsed with purified 18.2 MΩ deionized water and dried using gentle nitrogen gas flow. All samples were measured at room temperature in air. Structural analysis and height measurements of acquired images were performed with Nanoscope v.6.13 software (Watsonville, California, USA).

Molecular Weight Estimation with Gel Permeation Chromatography
The average molecular masses, Mn, Mw and dispersity Mw/Mn of the products were measured using gel permeation chromatography (GPC) using a RI detector (Shodex RI-71). The GPC instrument was equipped with TSKgel GMH HR -M and TSKgel GMH HR Guard column packed with styrene divinylbenzene-type gel. The measurements were performed at a temperature of 22 • C. All samples were dissolved in N,N-dimethylformamide (HPLC grade) containing 5 mmol/L LiCl. The flow rate of the carrier solvent was 1.00 mL/min. The sample injection volume was 100 µL. The average molecular masses and dispersity were determined using OmniSEC software (Dublin, Ireland). The G2, G3, and G4 PAMAM dendrimers bearing 46, 78, and 140 2,3-dihydroxyproyl substituents and averaged molecular weight: 6600, 12,681, and 24,575 Da, respectively for G2 46gl , G3 78gl , and G4 140gl were used for calibration. For details see Appendix A: Figure A2 and Table A2.

Dynamic Light Scattering Measurements
DLS measurements were performed using a Zetasizer nano ZS instrument. Measurements were made under back-scattering conditions (fixed scattering angle 173 • ). All dendrimers were dissolved in water at ca 1.0 mM concentration. The size of dendrimers and megamers were calculated in volume mode. The results are presented graphically ( Figure A3) and values of diameter [nm] are collected in Table A3. 3.4.5. Crystallographic Measurements X-ray diffraction data of 1 was collected on an Xcalibur diffractometer with Saphire detector (Mo-Kα radiation; λ = 0.71073 Å) at 100 K. Data reduction and analysis were carried out with the CrysAlis program [34]. Structure was solved by direct methods using the SHELXS program and refined using all F 2 data, as implemented by the SHELXL program [35]. Non-hydrogen atoms were refined with anisotropic displacement parameters. All H atoms were placed at calculated positions, and before the last cycle of refinement all H atoms were fixed and were allowed to ride on their parent atoms. The .cif file has been deposited in CCDC, number 1910753, and is available upon request via www.ccdc.cam.ac.uk.data/data_request.cif.

Conclusions
PAMAM G3 and G0 megamer composed of PAMAM G3 dendrimer with 64 terminal propyldiol substituents serving as a core and 12 PAMAM G0 subunits bearing carbonyl-linked nimesulide were obtained using p-nitrophenyl chloroformate as an efficient activator of both the hydroxyl group of the G3 core and nimesulide. The obtained conjugate is well soluble in water.
The megamer-nimesulide conjugate with G3 gl core and ca 12 G0 2N satellite dendrimers obtained was shown to effectively penetrate cells in micromolar concentrations. The conjugate has selective anticancer activity against human squamous carcinoma and glioma cells compared to normal human fibroblasts via inhibition of cell proliferation. Therefore, this megameric carrier provides a promising route for selective squamous carcinoma therapy. Additionally, megamers can be equipped with various drug and targeting molecules like biotin or folate, and other anticancer drugs that may additionally increase the effectiveness of anticancer therapy.

Conflicts of Interest:
The authors declare no conflicts of interest.
Appendix A Table A1. Bond lengths and torsion angles in 1.

Species Diameter [nm]
G2 gl 3.24 ± 0.11 G3 gl 3.94 ± 0.11 G4 gl 5.12 ± 0.12 G5 gl 6.35 ± 0.14 G3 G0N 5.07 ± 0.16 Figure A3. Size distribution of GM gl dendrimers and G3 G02N megamer (M-dendrimer generation). The PAMAM dendrimers coated with 2,3-dihydroxypropyl substituents were synthesized for these Figure A2. Molecular weight distribution curves of megamers G3 G0 and G3 G02N estimated using GPC experiments using the references obtained by reaction of PAMAM dendrimers G2, G3, and G4 with gl according to the protocol described for G3 gl . Obtained derivatives bear 46, 78, and 140 2,3-dihydroxyproyl substituents and averaged molecular weight 6600, 12,681, and 24,575 Da, respectively for G2 46gl , G3 78gl , and G4 140gl . The stoichiometry of these G2 gl , G3 gl , and G4 gl derivatives was determined using 1 H NMR spectroscopy. The G2 gl , G3 gl , and G4 gl were used as molecular weight standards to determine the molecular weight of megamers. The figure represents normalized weight fraction vs logM (where M-molecular weight). Samples of megamers G3 G0 a and G3 G0 b were obtained from G3 64gl : 18 NPCF : 20 G0 according to general protocol described in Experimental section. The average molecular weight of the samples G3 G0 b and G3 G0 a were estimated for conjugate before (b) and after (a) dialysis. Partial loss of G0 from the conjugate upon three days extensive dialysis can be concluded from that experiment (from average 13 to 10).  Figure A2. Molecular weight distribution curves of megamers G3 G0 and G3 G02N estimated using GPC experiments using the references obtained by reaction of PAMAM dendrimers G2, G3, and G4 with gl according to the protocol described for G3 gl . Obtained derivatives bear 46, 78, and 140 2,3dihydroxyproyl substituents and averaged molecular weight 6600, 12,681, and 24,575 Da, respectively for G2 46gl , G3 78gl , and G4 140gl . The stoichiometry of these G2 gl , G3 gl , and G4 gl derivatives was determined using 1 H NMR spectroscopy. The G2 gl , G3 gl , and G4 gl were used as molecular weight standards to determine the molecular weight of megamers. The figure represents normalized weight fraction vs logM (where M-molecular weight). Samples of megamers G3 G0 a and G3 G0 b were obtained from G3 64gl : 18 NPCF : 20 G0 according to general protocol described in Experimental section. The average molecular weight of the samples G3 G0 b and G3 G0 a were estimated for conjugate before (b) and after (a) dialysis. Partial loss of G0 from the conjugate upon three days extensive dialysis can be concluded from that experiment (from average 13 to 10). Table A2. Molecular weight parameters determined using GPC (see Figure A2 for description).  Figure A3. Size distribution of GM gl dendrimers and G3 G02N megamer (M-dendrimer generation).

Species
The PAMAM dendrimers coated with 2,3-dihydroxypropyl substituents were synthesized for these Figure A3. Size distribution of GM gl dendrimers and G3 G02N megamer (M-dendrimer generation). The PAMAM dendrimers coated with 2,3-dihydroxypropyl substituents were synthesized for these studies. The composition of G2 gl -G5 gl was determined using 1 H NMR spectroscopy. The NMR studies on the series will be reported separately.