Polyamidoamine Dendron-Bearing Lipids as Drug-Delivery Excipients

An amine-terminated polyamidoamine (PAMAM) dendron and two long alkyl groups were designed as a novel drug carrier that possesses an interior for the encapsulation of drugs and a biocompatible surface. We synthesized three dendron-bearing lipids, DL-G1, DL-G2, and DL-G3, which included first, second, and third generation polyamidoamine dendrons, respectively. The synthesized dendrimer encapsulating anticancer drug, 5-fluorouracil (5-FU), was prepared by extraction with chloroform from mixtures of the dendrimers and varying amounts of the drug. In vitro cytotoxicity of PAMAM conjugated di-n-dodecylamine micelles (G1, G2, G3) were analyzed on human gastric adenocarcinoma cells (AGS) by water-soluble tetrazolium-1 (WST-1) cell proliferation assay. Upon exposure to 5-FU loaded micelles, the viability of the cells decreased gradually in all generations. Cytotoxicity increased with increasing generation and reached its highest rate of 69.8 ± 3.2% upon 15 µM 5FU-loaded 25 µM PAMAM DL-3 micelle treatment. These results demonstrate that 5FU-loaded PAMAM conjugated di-n-dodecylamine treatment inhibits the proliferation of AGS cells in a generation-dependent manner.


Introduction
Cancer is a leading cause of death worldwide and is responsible for approximately 13% of all deaths, according to the World Health Organization. Chemotherapy is an essential component in the multidisciplinary management of most cancers. The anticancer drug 5-FU is widely used for the treatment of a wide range of cancer types, including colorectal and breast cancers and cancers of the aerodigestive tract [1]. It is a BCS class III drug that shows good water solubility and poor permeability; i.e., it is a hydrophilic drug having 12.20 mg ml −1 [2]. Due to the high toxicity of the drug, it cannot generally be used in adequate doses and/or adequate periods of treatment. Therefore, 5-FU started to be used with other chemotherapeutic agents to increase the therapeutic index. Nonetheless, the response rates for 5-FU-based chemotherapy as a first-line treatment for advanced colorectal cancer are only in the range of 10-15% [3]. The combination of 5-FU with newer chemotherapeutics, such as irinotecan and oxaliplatin, has improved the response rates for advanced colorectal cancer to 40-50% [3].
The other major problem with the vast majority of clinically used drugs is their short half-life in the bloodstream and their high overall clearance rate. A relatively small amount of the drug reaches the target site to provide the desired reaction, while the non-selective distribution in the body gives undesired reactions and leads to side effects [4]. For these 76.6 ±0.8, respectively [20]. Solid lipid nanoparticle (SLN) formulae were also utilized for the release of 5-FU inside the colonic medium for local treatment of colon cancer. SLNs were prepared by a double-emulsion-solvent evaporation technique (w/o/w) using triglyceride esters, Dynasan™ 114, or Dynasan™ 118, along with soyalecithin as the lipid part [21]. The 5-FU was also encapsulated into magnetite-zeolite nanocomposite particles in our previous study [22]. The cCytotoxic effects of 5-FU-loaded MZNC on human gastric carcinoma (AGS) cells were determined by real-time cell analysis and colorimetric WST-1 cell viability assay.
The aim of the study was to investigate the micelle formation and toxicity of dendronbearing lipids with PAMAM G1, G2, and G3 dendrons. For this reason, a novel cationic amphiphilic lipid with a hydrophilic tail and hydrophobic core was initially synthesized. Then, PAMAM dendrimers were converted to micelles with the ability to carry waterinsoluble drugs in their hydrophobic core. For use as an anti-cancer agent, 5-FU was loaded into PAMAM dendrons of first to third generation, DL-G1, DL-G2, and DL-G3. Finally, the in vitro cytotoxicity of PAMAM dendrimers in all generations, with or without 5-FU, was analyzed on AGS cells.

Characterization
In the FT-IR analysis of the dendrimers, several characteristic picks were determined in Figure 1. Because of the nature of the dendrimers, two different reactions were used for building the generations-esterification followed by amination-generally referred to as Michael addition. As only side groups can provide strong peaks in bulky materials such as dendrimers, to confirm those reactions, the signals of the ester and amine groups were checked in every step of the reaction by FT-IR ( Figure 1). The FTIR spectra of the full generation dendrimers were of N-H stretch for terminal primary amine at averages of 3330 cm −1 , 3284 cm −1 , and 3245 cm −1 and C-N stretch of primary amine at averages of 1312 cm −1 and 1280 cm −1 . With increasing generations, the N-H number increased and peaks were seen at higher wavelengths. IR spectra of half generation of dendrimers were of C=O stretch of the carbonyl group; peaks were at an average of 1732 cm −1 .
Molecules 2022, 27, x FOR PEER REVIEW 3 of 12 study the impact on the "ghosts" cell wall. 5-FU was then loaded into the bacterial ghosts and the loading capacity and the entrapment efficiency were determined; they were found to be 38.3 ± 0.8 and 76.6 ±0.8, respectively [20]. Solid lipid nanoparticle (SLN) formulae were also utilized for the release of 5-FU inside the colonic medium for local treatment of colon cancer. SLNs were prepared by a double-emulsion-solvent evaporation technique (w/o/w) using triglyceride esters, Dynasan™ 114, or Dynasan™ 118, along with soyalecithin as the lipid part [21]. The 5-FU was also encapsulated into magnetite-zeolite nanocomposite particles in our previous study [22].The cCytotoxic effects of 5-FU-loaded MZNC on human gastric carcinoma (AGS) cells were determined by real-time cell analysis and colorimetric WST-1 cell viability assay. The aim of the study was to investigate the micelle formation and toxicity of dendronbearing lipids with PAMAM G1, G2, and G3 dendrons. For this reason, a novel cationic amphiphilic lipid with a hydrophilic tail and hydrophobic core was initially synthesized. Then, PAMAM dendrimers were converted to micelles with the ability to carry waterinsoluble drugs in their hydrophobic core. For use as an anti-cancer agent, 5-FU was loaded into PAMAM dendrons of first to third generation, DL-G1, DL-G2, and DL-G3. Finally, the in vitro cytotoxicity of PAMAM dendrimers in all generations, with or without 5-FU, was analyzed on AGS cells.

Characterization
In the FT-IR analysis of the dendrimers, several characteristic picks were determined in Figure 1. Because of the nature of the dendrimers, two different reactions were used for building the generations-esterification followed by amination-generally referred to as Michael addition. As only side groups can provide strong peaks in bulky materials such as dendrimers, to confirm those reactions, the signals of the ester and amine groups were checked in every step of the reaction by FT-IR ( Figure 1). The FTIR spectra of the full generation dendrimers were of N-H stretch for terminal primary amine at averages of 3330 cm −1 , 3284 cm −1 , and 3245 cm −1 and C-N stretch of primary amine at averages of 1312 cm −1 and 1280 cm −1 . With increasing generations, the N-H number increased and peaks were seen at higher wavelengths. IR spectra of half generation of dendrimers were of C=O stretch of the carbonyl group; peaks were at an average of 1732 cm −1 .

CMC Analysis
The critical micelle concentration of the new amphiphiles was in range of 2.5 × 10 −6 to 5.0 × 10 −6 (Table S1). Thus, the minimum concentration of amphiphiles for the formation of micelles was remarkably low. This may be beneficial with regard to formulation stability, especially after dilution prior to or after intravenous application. As a result, CMC was determined as DL-G1: 2.5 × 10 −6 , DL-G2: 3.7 × 10 −6 , DL-G3: 5.0 × 10 −6 .

Drug Loading Studies
The effect of micelle concentration on the solubility of 5-FU was measured at 37 • C and the results are shown in the Figure 2. It was observed that the solubility of 5-FU was approximately 2.8 to 15 times better than that of water. For higher generations of dendrimer, the solubility of 5-FU was increased, as expected, but with the increasing concentrations, the solubility increase was less than expected. This might have been due to newly forming micelles with increases in concentration. This might also have been due to the solubilization process itself, which is a very slow distribution process. Nevertheless, a high solubilization of the poor water-soluble drug was achieved with this method, in the absence of any organic solvent and heat.
The critical micelle concentration of the new amphiphiles was in range of 2.5 5.0 × 10 −6 (Table S1). Thus, the minimum concentration of amphiphiles for the for of micelles was remarkably low. This may be beneficial with regard to form stability, especially after dilution prior to or after intravenous application. As a CMC was determined as DL-G1: 2.5 × 10 −6 , DL-G2: 3.7 × 10 −6 , DL-G3: 5.0 × 10 −6 .

Drug Loading Studies
The effect of micelle concentration on the solubility of 5-FU was measured a and the results are shown in the Figure 2. It was observed that the solubility of 5approximately 2.8 to 15 times better than that of water. For higher generat dendrimer, the solubility of 5-FU was increased, as expected, but with the inc concentrations, the solubility increase was less than expected. This might have be to newly forming micelles with increases in concentration. This might also have b to the solubilization process itself, which is a very slow distribution process. Never a high solubilization of the poor water-soluble drug was achieved with this met the absence of any organic solvent and heat.
According to the results, cell proliferation was decreased in a concen dependent manner in all generations. While cells at 10 and 25 µM concentration near to the control, micelles at higher concentrations reduced cell proliferation. T revealed that 50 µM and higher concentrations were significantly cytotoxic generations (p < 0.05, Figure 3). It is known that the toxicity of dendrimers is mainly to the end groups. Generally, amine-terminated PAMAM dendrimers concentration-dependent toxicity, which increases with generations [8,11,23]. treatment with micelles at elevated doses reduced the cell proliferation, the concentration, which has a greater drug-loading capacity than 10 µM (as ther significant difference in toxicity between 10 and 25 µM) was used in further expe during the study.
According to the results, cell proliferation was decreased in a concentration-dependent manner in all generations. While cells at 10 and 25 µM concentrations were near to the control, micelles at higher concentrations reduced cell proliferation. The data revealed that 50 µM and higher concentrations were significantly cytotoxic in all generations (p < 0.05, Figure 3). It is known that the toxicity of dendrimers is mainly related to the end groups. Generally, amine-terminated PAMAM dendrimers display concentration-dependent toxicity, which increases with generations [8,11,23]. As the treatment with micelles at elevated doses reduced the cell proliferation, the 25 µM concentration, which has a greater drugloading capacity than 10 µM (as there is no significant difference in toxicity between 10 and 25 µM) was used in further experiments during the study. In order to assess the IC50 of 5-FU on AGS cells, elevating concentrations in the range of 1 to 100 µM 5-FU were applied to the cells, and the inhibition of cell proliferation was measured for 24 h using the WST-1 assay. According to the results, 5-FU showed significantly toxic effects in all concentrations upon its administration on AGS cells. The IC50 value of 5-FU for AGS cells was determined to be 15 µM, exhibiting 50.1 ± 1.7% cell death in 24 h (p < 0.05; Figure 4). Therefore, micelles were loaded with 15 µM 5-FU for further experiments. In order to assess the IC50 of 5-FU on AGS cells, elevating concentrations in the range of 1 to 100 µM 5-FU were applied to the cells, and the inhibition of cell proliferation was measured for 24 h using the WST-1 assay. According to the results, 5-FU showed significantly toxic effects in all concentrations upon its administration on AGS cells. The IC50 value of 5-FU for AGS cells was determined to be 15 µM, exhibiting 50.1 ± 1.7% cell death in 24 h (p < 0.05; Figure 4). Therefore, micelles were loaded with 15 µM 5-FU for further experiments. In order to assess the IC50 of 5-FU on AGS cells, elevating concentrations in the range of 1 to 100 µM 5-FU were applied to the cells, and the inhibition of cell proliferation was measured for 24 h using the WST-1 assay. According to the results, 5-FU showed significantly toxic effects in all concentrations upon its administration on AGS cells. The IC50 value of 5-FU for AGS cells was determined to be 15 µM, exhibiting 50.1 ± 1.7% cell death in 24 h (p < 0.05; Figure 4). Therefore, micelles were loaded with 15 µM 5-FU for further experiments.  AGS cells were exposed to 15 µM of 5-FU-loaded G1, G2, and G3 di-n-dodecylamine micelles for 24 h, and the percentage of cell viability was assessed by WST-1 assay.
Treatment with the drug-loaded form of micelles exhibited remarkably more cytotoxicity than treatment with the drug itself. While 15 µM 5-FU induced 50.1 ± 1.7% cell death, 5-FU-loaded DL-G1 caused higher toxicity, with 59.0 ± 6.5% cell death in 24 h.
Micelles are composed of lipids similar to those present in biological membranes and, therefore, they are readily accepted into the cells via endocytosis [16]. Our results suggest that the micelles facilitate the entry of the drug into the cell; thus, higher toxicity was observed in the drug-loaded micelles.
Moreover, a generation-dependent gradual decline in cell viability was observed among 5-FU-loaded micelles. The 5-FU-loaded G1 and G2 micelles showed significant cytotoxicity on AGS cells, inducing 59.0 ± 6.5% and 63.7 ± 3.2% cell death, compared with control cells(p < 0.05, Figure 5). Di-n-dodecylamine micelles in the third and highest generation (G3) significantly inhibited cell proliferation, demonstrating 69.1 ± 7.3% cell death and exhibiting a significant antiproliferative effect, compared with G1 micelles (p < 0.05, Figure 5). The interaction between negatively charged cell membranes and the increased positively charged dendrimer surface can explain why increased generations of cationic dendrimers are more cytotoxic. This increased interaction caused the cell membrane to be damaged, causing nanopores to develop, which ultimately caused the cell to die. At the same time, as the generation increased, the drug-carrying capacity of the denrimer increased and caused more cellular death [24]. To assess the IC50 of 5-FU, cells were exposed to 2.5, 5, 10, 25, 50, and 100 µM 5FU for 24 h, and cell viability was evaluated by WST-1 assay. The percentage of cell viability relative to untreated (control) cells (A) and IC50 value of 5-FU (B) were presented in the graphs. Error bars represent the standard deviation of the mean (* p < 0.05).
AGS cells were exposed to 15 µM of 5-FU-loaded G1, G2, and G3 di-n-dodecylamine micelles for 24 h, and the percentage of cell viability was assessed by WST-1 assay.
Treatment with the drug-loaded form of micelles exhibited remarkably more cytotoxicity than treatment with the drug itself. While 15 µM 5-FU induced 50.1 ± 1.7% cell death, 5-FU-loaded DL-G1 caused higher toxicity, with 59.0 ± 6.5% cell death in 24 h.
Micelles are composed of lipids similar to those present in biological membranes and, therefore, they are readily accepted into the cells via endocytosis [16]. Our results suggest that the micelles facilitate the entry of the drug into the cell; thus, higher toxicity was observed in the drug-loaded micelles.
Moreover, a generation-dependent gradual decline in cell viability was observed among 5-FU-loaded micelles. The 5-FU-loaded G1 and G2 micelles showed significant cytotoxicity on AGS cells, inducing 59.0 ± 6.5% and 63.7 ± 3.2% cell death, compared with control cells(p < 0.05, Figure 5). Di-n-dodecylamine micelles in the third and highest generation (G3) significantly inhibited cell proliferation, demonstrating 69.1 ± 7.3% cell death and exhibiting a significant antiproliferative effect, compared with G1 micelles (p < 0.05, Figure 5). The interaction between negatively charged cell membranes and the increased positively charged dendrimer surface can explain why increased generations of cationic dendrimers are more cytotoxic. This increased interaction caused the cell membrane to be damaged, causing nanopores to develop, which ultimately caused the cell to die. At the same time, as the generation increased, the drug-carrying capacity of the denrimer increased and caused more cellular death [24]. Figure 5. Evaluation of the cytotoxic activity of 5FU-loaded PAMAM-conjugated di-ndodecylamine micelles (DL-G1, DL-G2, DL-G3). AGS cells were exposed to 15 µM 5FU-loaded 25 µM DL-G1, DL-G2, and DL-G3 micelles for 24 h, and cell viability was assessed by WST-1 assay. Data were presented as the percentage of cell viability relative to the control group of each generation. Error bars represent the standard deviation of the mean (* p < 0.05).
Taken together, these data suggest that 5-FU loaded G1, G2, and G3 di-ndodecylamine micelles possess anti-proliferative activity and induce cell death in a generation-dependent manner, and especially that G3 micelles significantly increase cell death, compared with G1. Figure 5. Evaluation of the cytotoxic activity of 5FU-loaded PAMAM-conjugated di-n-dodecylamine micelles (DL-G1, DL-G2, DL-G3). AGS cells were exposed to 15 µM 5FU-loaded 25 µM DL-G1, DL-G2, and DL-G3 micelles for 24 h, and cell viability was assessed by WST-1 assay. Data were presented as the percentage of cell viability relative to the control group of each generation. Error bars represent the standard deviation of the mean (* p < 0.05).

Materials and Methods
Taken together, these data suggest that 5-FU loaded G1, G2, and G3 di-n-dodecylamine micelles possess anti-proliferative activity and induce cell death in a generation-dependent manner, and especially that G3 micelles significantly increase cell death, compared with G1.

Chemicals and Reagents
All chemicals and solvents were reagent grade and were used as purchased without further purification. Electronic spectral studies were conducted on a Shimadzu 2850 model UV-VIS spectrophotometer. ATR spectra were recorded as solid or liquid on Bruker Alpha-P in the range of 4000-400 cm −1 . Routine 1 H (400 MHz) and 13 C (100 MHz) spectra were recorded in 13 CDC at ambient temperature on a Bruker Ultrashield Plus 400 MHz instrument. Chemical shifts (δ) were expressed in units of parts per million relative to TMS. Micelle images were studied on HR-SEM. The analytical data, mass, ATR, NMR, and physical properties were summarized for each experiment.

Synthesis of PAMAM Dendron Bearing Lipids
A series of polyamidoamine dendron-bearing lipids were synthesized by repetition of exhaustive Micheal addition with methyl acrylate using di-n-dodecylamine as the starting material and subsequent exhaustive amidation with ethylenediamine, as reported by Tomalia [25][26][27]. The synthetic route for the polyamidoamine dendron-bearing lipids is shown in Figure 6.

Chemicals and Reagents
All chemicals and solvents were reagent grade and were used as purchased without further purification. Electronic spectral studies were conducted on a Shimadzu 2850 model UV-VIS spectrophotometer. ATR spectra were recorded as solid or liquid on Bruker Alpha-P in the range of 4000-400 cm −1 . Routine 1 H (400 MHz) and 13 C (100 MHz) spectra were recorded in 13 CDC at ambient temperature on a Bruker Ultrashield Plus 400 MHz instrument. Chemical shifts (δ) were expressed in units of parts per million relative to TMS. Micelle images were studied on HR-SEM. The analytical data, mass, ATR, NMR, and physical properties were summarized for each experiment.

Synthesis of PAMAM Dendron Bearing Lipids
A series of polyamidoamine dendron-bearing lipids were synthesized by repetition of exhaustive Micheal addition with methyl acrylate using di-n-dodecylamine as the starting material and subsequent exhaustive amidation with ethylenediamine, as reported by Tomalia [25][26][27]. The synthetic route for the polyamidoamine dendron-bearing lipids is shown in Figure 6.

Preparation of PAMAM Dendron Lipid Containing Liposomes
Micelles were made by direct dissolution of amphiphilic DL-G1, DL-G2, and DL-G3 dendron lipids into PBS (pH = 7.4) at 50 • C for 5 min. in 0.1, 0.5, 1, and 2 mM concentrations. The icelle size and the poldispersity index (PDI) were determined by a scanning electron microscope (SEM). The samples for the SEM were prepared by dropping a few drops of the micelle solution on a thin layer and desiccating them in a desiccator.

Characterisation of Liposomes
The 5-FU was loaded into micelles by the excess method. One mL of DL-G1, DL-G2, and DL-G3 solutions in 0.1, 0.5, 1, and 2 mM concentrations were put into Eppendorf tubes; then, 100 mg of 5-FU was added to each. The tubes were put into a shaker for 3 h at 37 • C. After that, 100 mg more 5-FU was added to the ones that were fully or nearly dissolved and put back into the shaker for 24 h at 37 • C. The undissolved drug and the micelle solutions were separated by using syringe filters with 0.24 µm pore size [6]. The drug content was determined by a UV-Vis spectrometer, by analyzing the peaks at 265 nm.

Determination of Critical Micelle Concentration (CMC)
In order to determine the CMC, UV-Vis spectrophotometry was used. The micelles were determined at 221 nm wavelength ( Figure S1). Starting from 1 mM, the micelle solution was diluted until the peak disappeared. As CMC defines the concentration at which micelles are formed, it is also equal to the concentration at which micelles disappear [28].
3.6. In Vitro Assays 3.6.1. Cell Culture AGS (CRL-1739) cells were purchased from the American Type Culture Collection (ATCC) company, thawed, passaged, and stored in liquid nitrogen for further experiments. The cells were subcultured in DMEM containing 10% FBS and 0.1 mg/mL penicillinstreptomycin and incubated in a humidified atmosphere of 5% CO 2 at 37 • C. The medium was refreshed every 48 h and the cells were passaged upon 70-80% confluency for continuous culture.

Cytotoxicity Assay
The cytotoxic activity of 5-FU-and PAMAM-conjugated di-n-dodecylamine (G1, G2, G3) micelles on AGS cells with and without 5-FU was tested using the WST-1 cell proliferation assay. WST-1 was used to evaluate cell viability according to mitochondrial dehydrogenase activity, which cleaves the tetrazolium salt WST-1 into formazan dye. The WST-1 assay was performed according to the manufacturer's instructions. The cells were seeded into triplicate 96 well plates at a density of 1.5 × l0 4 cells/well and incubated overnight. Initially, to determine the IC50 value of 5-FU, AGS cells were exposed to various concentrations of 5-FU (1, 2.5, 5, 10, 25, 50 µM) for 24 h. Second, to evaluate the cytotoxicity of the micelles themselves, the cells were treated with various concentrations of G1, G2, and G3 micelles (10, 25, 50, 100, 250, and 500 µM) for 24 h. Finally, to analyze the antiproliferative activity of 5-FU-loaded micelles in all generations, the cells were treated with 15 mM 5-FU-loaded 25 µM DL-G1, DL-G2, and DL-G3 micelles for 24 h. After incubation, WST-1 solution was added to the cells and incubated for 4 h. Finally, absorbance was read at 450 nm using a microplate reader (Synergy H1, Biotek).

Statistical Analyses
Data were evaluated by a one-way ANOVA test and an unpaired Student's t-test using the SPSS 25 program. All of the experiments were performed in triplicates and the results were presented as mean ± standard deviation (SD). p values less than 0.05 (* p < 0.05) were considered as statistically significant.

Conclusions
The anticancer drug 5-FU has a restricted bioavailability because of its lower solubility and low bioavailability In this work, PAMAM bearing dendron lipids with three different generations have been discussed as for drug-delivery application. It was observed from the phase solubility studies that the solubility of 5-FU increased proportionally with the increasing amount of added dendrimer concentrations. Also, the results showed that their ability to encapsulate these drugs increased with increasing dendrimer generation. Moreover, 5FU-loaded PAMAM conjugated DL micelles significantly inhibit cell proliferation of AGS cells and exhibited cytotoxic activity which increases with generation. The utilization of drug-loaded dendritic micelles is a promising approach to overcome the high systemic toxicity and low solubility of conventional chemotherapy drugs. Further studies are encouraged to evaluate the role of DL micelles as a drug vehicle in various types of tumors in animal models.