RSC Advances PAPER View Article Online View Journal | View Issue Efficient modification of PAMAM G1 dendrimer surface with b-cyclodextrin units by CuAAC: impact Cite this: RSC Adv., 2020, 10, 25557 on the water solubility and cytotoxicity† a a Kendra Sorroza-Mart´ınez,‡ Israel Gonzalez-M´ endez,´ ‡ Ricardo D. Mart´ınez- a b a c d Serrano, Jose´ D. Solano, Andrea Ruiu, Javier Illescas, Xiao Xia Zhu and Ernesto Rivera *a The toxicity of the poly(amidoamine) dendrimers (PAMAM) caused by the peripheral amino groups has been a limitation for their use as drug carriers in clinical applications. In this work, we completely modified the periphery of PAMAM dendrimer generation 1 (PAMAM G1) with b-cyclodextrin (b-CD) units through the Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) to obtain the PAMAM G1-b-CD dendrimer with high yield. The PAMAM G1-b-CD was characterized by 1H- and 13C-NMR and mass spectrometry studies. Moreover, the PAMAM G1-b-CD dendrimer showed remarkably higher water solubility than native b-CD. Received 20th March 2020 Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Finally, we studied the toxicity of PAMAM G1-b-CD dendrimer in four different cell lines, human breast Accepted 30th June 2020 cancer cells (MCF-7 and MDA-MB-231), human cervical adenocarcinoma cancer cells (HeLa) and pig DOI: 10.1039/d0ra02574g kidney epithelial cells (LLC-PK1). The PAMAM G1-b-CD dendrimer did not present any cytotoxicity in cell rsc.li/rsc-advances lines tested which shows the potentiality of this new class of dendrimers. Introduction ethylenediamine or ammonia, repetitive units of polyamides, tertiary amines and a periphery containing primary amines.4 It In the last decades, research on the development of new den- has been found that low generations (G 0–3) of PAMAM den- This article is licensed under a drimers has become more relevant and attracted the attention drimers tend to form open and amorphous structures, and of the scientic community, since these compounds have therefore do not exhibit well dened internal characteristics, unique properties that make them ideal carriers for therapeutic while high generations dendrimers (G 4–10) adopt spherical Open Access Article. Published on 07 July 2020. Downloaded 9/29/2021 8:04:03 AM. and diagnostic agents in the construction of drug delivery and rigid structures that allow them to encapsulate drug systems (DDS).1 One advantage of dendrimers is that between molecules in their branches.5,6 the small molecules and the polymers, dendrimers benet from At physiological conditions (pH ¼ 7.4) the primary amines of the properties of both elds. Given the versatility to modify the PAMAM dendrimers are positively charged,7 they exhibit them (like polymers), it is possible to guarantee a perfectly toxicity, since they interact with the cell membrane, which is dened and reproducible structure.2 It has been described that negatively charged, compromising its integrity and ltering the groups in the periphery are largely responsible for the intracellular components, that nally lead to a cell death. This physicochemical properties of dendrimers and can be modied toxicity is also associated with the increase in generation, due to in a controlled manner to yield monodisperse materials.3 the augment in the number of terminal amino groups. For this One of the most studied dendrimers, is the family of poly(- reason, the clinical application of PAMAM dendrimers is quite amidoamines) (PAMAM), which is constituted by a core of limited.8,9 In recent years, research has focused on improving the biocompatibility of PAMAM dendrimers by surface engineering, aInstituto de Investigaciones en Materiales, Universidad Nacional Autonoma´ de that means the modication of the periphery to neutralize the M´exico, Circuito Exterior, Ciudad Universitaria, CP 04510, M´exico City, M´exico. E-mail: [email protected] primary amines that are positively charged on the surface, bFacultad de Qu´ımica, Departamento de Biolog´ıa, Universidad Nacional Autonoma´ de thereby preserving the ability to deliver drugs. There are several M´exico, Circuito Exterior, Ciudad Universitaria, CP 04510, M´exico City, M´exico strategies to modify the periphery of PAMAM dendrimers, such cTecnologico´ Nacional de M´exico/Instituto Tecnologico´ de Toluca, Av. Tecnologico´ S/N, as coupling with carbohydrates, amino acids or peptides, acet- Col. Agr´ıcola Bellavista CP 52149, Metepec, M´exico ylations and PEGylations.8 One of the strategies used in surface d D´epartement de Chimie, Universit´e de Montr´eal, C.P. 6128, Succursale Centre-ville, engineering of PAMAM dendrimers is the coupling with cyclo- Montreal, QC, H3C 3J7, Canada dextrins because it increases the solubility, stability and † Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ra02574g biocompatibility of dendrimers and therefore causes a syner- 10 ‡ These people contributed equally to this work. gistic improvement in the administration of drugs. This journal is © The Royal Society of Chemistry 2020 RSC Adv.,2020,10, 25557–25566 | 25557 View Article Online RSC Advances Paper Chemically, cyclodextrins (CDs) are cyclic oligosaccharides the in vitro cellular behavior of this novel dendrimer as part of linked through a-1,4 glucosidic bonds. The nomenclature of the the rational evaluation, since our perspective is based on the CDs is based on the number of glucose units contained in their potential use of this PAMAM G1-b-CD conjugate as a possible structure, so that the CDs containing six units are called alpha- carrier of diverse molecules. The advantage of this new modi- CD (a-CD), seven units, beta-CD (b-CD) and eight units, gamma- ed dendrimer is that it is able to carry a higher number of drug CD (g-CD).11 As a result of the restricted rotation of the bonds in units than a single carrier. the glucose units by the chair conformation, CDs molecules have a truncated cone shape with a cavity of determined Results and discussion volume. The hydroxyl groups attached to the carbons 3 and 4 are located on the secondary face, and the hydroxyl groups attached Synthesis to carbon 6 are located on the primary face of the CDs; these In recent years, numerous reports described the advantages of hydroxyl groups have a different reactivity, because they are PAMAM dendrimers in various areas of science and technology; attached to secondary and primary carbons, respectively. The however, there are also some disadvantages that limit their use surface of the CDs is coated with hydroxyl groups and the inner in clinical applications. Firstly, PAMAM dendrimers are toxic part is coated with ether groups of anomeric oxygen atoms. and this toxicity can be only resolved by modifying the structure Therefore, CDs have a relatively hydrophobic internal cavity, of the periphery of the dendrimer.33 Intense research has which contains water molecules and a hydrophilic outer focused on the surface engineering of PAMAM dendrimers with surface. This special conformation allows the inclusion of the aim of reducing or eliminating their toxicity by neutralizing hydrophobic molecules or parts of molecules within the CD the amino groups in the periphery by means of coupling of cavity, which must have a compatible size with the cavity of CD, neutral or anionic groups in order to prevent the electrostatic to form a stable inclusion complex.12 b-CD is the most acces- interaction of PAMAM dendrimers with biological membranes sible and widely used CD in the design of DDS, due to the to help them to be biocompatible for their use as drug carriers.8 Creative Commons Attribution-NonCommercial 3.0 Unported Licence. adequate size of its cavity to form inclusion complexes with A wide variety of methods have been reported to modify the drug molecules.13 surface of PAMAM dendrimers, including modication with On the other hand, the 1,3-dipolar cycloaddition reaction carbohydrates, specically with oligosaccharides such as CDs, between an azide and a terminal alkyne catalyzed by copper(I) since it has been reported that a synergy of the properties of (CuAAC) which produces 1,4-disubstituted 1,2,3-triazoles was PAMAM dendrimers and CDs occurs.34–36 PAMAM and b-CD reported independently in 2001 by Meldal et al.14 and Sharpless dendrimers have been individually investigated as promising et al.15,16 Meldal used copper iodide and N,N-diisopropylethyl- carriers for clinical applications; however, due to the toxicity of amine for the synthesis of peptides, while Sharpless described PAMAM dendrimers8,37 and the low water solubility of b-CD,38 the use of copper sulfate pentahydrate and sodium ascorbate in their application in the clinical area is limited. In the present This article is licensed under a aqueous systems and designed a practical procedure for the study we present a novel and efficient approach to modify the covalent attachment between any molecule containing an azide periphery of the PAMAM G1 dendrimer with b-CD units through group and another molecule containing a terminal alkyne the CuAAC reaction. All the intermediates and nal compounds Open Access Article. Published on 07 July 2020. Downloaded 9/29/2021 8:04:03 AM. group. The reaction of CuAAC is modular, specic and regio- involved in the synthesis have been fully characterized. selective, inert byproducts are obtained and offers high yields. Firstly, alkynyl-PAMAM G1 was synthesized (Scheme l) and In addition, this reaction is carried out under simple conditions was used as alkyne source for the CuAAC reaction. A Williamson and with accessible raw materials.17 During the last decade, the etherication reaction was carried out between propargyl CuAAC reaction has proven to be a reliable and versatile tech- bromide and methyl 2-(4-hydroxyphenyl)acetate, to obtain nique for the preparation of new functional materials,18,19 intermediate I, whose structure was conrmed by 1H-NMR and including dendrimers as well as the modication of their 13C-NMR and by ESI mass spectrometry (Fig.