therapy

Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1481 – 1498

Review Article

nanomedjournal.com

Polyanionic carbosilane dendrimer-conjugated antiviral drugs as efficient microbicides: Recent trends and developments in HIV treatment/therapy Daniel Sepúlveda-Crespo, PhDc a, b , Rafael Gómez, PhD c , Francisco Javier De La Mata, PhD c , José Luis Jiménez, PhD b,⁎, Mª. Ángeles Muñoz-Fernández, PhD, MD a, b,⁎ a Laboratorio InmunoBiología Molecular, IISGM, BioBank VIH HGM, CIBER-BBN, Madrid, Spain Plataforma de Laboratorio, Hospital Gregorio Marañón, IISGM, BioBank VIH HGM, CIBER-BBN, Madrid, Spain c Departamento de Química Inorgánica, Universidad de Alcalá, Campus Universitario, Alcalá de Henares, CIBER-BBN, Madrid, Spain Received 20 November 2014; accepted 19 March 2015 b

Abstract Polyanionic carbosilane dendrimers (PCDs) are potential candidates for the development of new microbicides for the prevention of HIV transmission. Tenofovir (TFV), which has dual antiviral activity (anti-HIV/HSV-2), and maraviroc (MRV) are the most studied antiretrovirals as microbicides. Here, we introduce developments in the design of innovative dendrimer-based microbicides. We also review and discuss the combination of various PCDs with TFV and/or MRV for their anti-HIV-1 activity and synergistic combinatory potential. Well-defined combinations blocking HIV-1 infection in early steps of HIV-1 replication provide greater efficacy than monotherapy, as reflected by the decrease in concentration and increase in HIV-1 inhibition. These combinations are characterized by lower doses, which minimize toxic side-effects and the emergence of multi-drug resistant mutants. The above facts suggest that the combination of first- and second-generation PCDs with TFV and/or MRV represents a promising candidate microbicide for preventing HIV-1 sexual transmission and simultaneously suppressing HSV-2. From the Clinical Editor: HIV infection remains a significant and unresolved problem for humankind, despite the development of combination antiretroviral therapy. It has been found that polyanionic carbosilane dendrimers have efficacy in preventing HIV transmission. In this comprehensive review article, the authors discuss the current status and latest development of the use of dendrimers in combination with other antiretroviral drugs as microbicides, which should stimulate others into further research in the fight against HIV. © 2015 Elsevier Inc. All rights reserved. Key words: HIV-1/HSV-2; Maraviroc; Microbicide; Polyanionic carbosilane dendrimers; Tenofovir

Despite continuing advances in the treatment and prevention of human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS), the HIV/AIDS pandemic currently represents an important and unsolved problem for most developed and developing countries. The absence of a cure or agents for stopping HIV-1 infection emphasizes the need for seeking out new approaches for HIV/AIDS treatment and prevention. The introduction of combination antiretroviral therapy (cARTs) has resulted in noteworthy successes for controlling

infection and reducing transmission and for significantly improving the average survival and quality of life for patients. 1 However, the emergence of viral resistance, life-long administration, poor patient adherence, short- and long-term side effects, and high costs that impede their widespread use in the developing world 2 make prevention an important strategy for impeding the epidemic. Sub-Saharan Africa is the region most affected by the HIV-1 spread, 3 where sexual contact is the primary route of HIV transmission. 4 HIV-1 infection in the female reproductive tract involves three major events: entry

Conflicts of Interest: The authors do not have a commercial or other association that might pose a conflict of interest. Source of Funding: This work has been (partially) funded by the RD12/0017/0037, project as part of the Plan Nacional R + D + I and cofinanced by ISCIII-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER), RETIC PT13/0010/0028, FIS (PI11/00888; PI13/02016; PI14/00882), CTQ2011-23245 (MIMECO), FIPSE, CAM (S-2010/BMD-2351; S-2010/BMD-2332), PENTA, CYTED 214RT0482 and the “Programa de Investigación de la Consejería de Sanidad de la CAM” to JLJ. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, the Consolider Program, and CIBER Actions and financed by the ISCIII with assistance from the European Regional Development Fund. ⁎Corresponding authors at: Laboratorio InmunoBiología Molecular; Plataforma de Laboratorio, Hospital G.U. Gregorio Marañón. CIBER BBN. IISGM, Madrid, Spain. E-mail addresses: [email protected] (J.L. Jiménez), [email protected] (Mª. Á. Muñoz-Fernández). http://dx.doi.org/10.1016/j.nano.2015.03.008 1549-9634/© 2015 Elsevier Inc. All rights reserved. Please cite this article as: Sepúlveda-Crespo D, et al, Polyanionic carbosilane dendrimer-conjugated antiviral drugs as efficient microbicides: Recent trends and developments in HIV treatment/therapy. Nanomedicine: NBM 2015;11:1481-1498, http://dx.doi.org/10.1016/j.nano.2015.03.008

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through the mucosal epithelium, productive infection in subepithelial mononuclear cells, and delivery to lymph nodes to initiate systemic infection. 5 The vulnerability of women to HIV-1 infection has increased due to cultural/social aspects (religion or polygamy), which do not provide women the power to negotiate the use of a condom, discuss fidelity with their partners or leave risky relationships. 6,7 In the absence of a feasible vaccine and the lack of effective therapies for HIV-1, 8 strategies focused on the use of longlasting and self-applied microbicides, which can be used vaginally or rectally by women or men, to halt the spread of HIV-1 during sexual intercourse are particularly needed in the underdeveloped countries. 9 The lack of effectiveness of surfactants, such as nonoxynol-9, and SAVVY (C31G), 10,11 and non-specific polyanions, such as cellulose sulfate, carraguard (carrageenan), and PRO2000, 12 leads to the urgent need for more stringent pre-clinical protocols and more research and progress for designing better systems based, for instance, on nanotechnology. 13,14 Nanotechnology and the various nanosystems used as HIV-1 therapeutics offer some advantages: bioavailability, stability, water solubility, low cost, and targeting the action of antiretrovirals (ARVs). 15,16 One of the most promising targets of the HIV-1 cycle is the viral entry-fusion process. Dendrimers containing several types of functionalized groups at their periphery have shown effective anti-HIV-1 activity as nonspecific microbicides because they can bind to their target in a multivalent manner and overcome weak monovalent interactions, providing a strategy for the development of viral entry inhibitors. 17

Microbicides in combination The function of microbicides is to protect against HIV-1 infection, possibly acting at multiple stages of HIV-1 replication. Thus, pre-exposure prophylaxis (PrEP) of HIV-1 transmission by microbicides needs to be combined with different classes of non-specific compounds or ARVs with non-specific compounds with two or more diverse mechanisms of action directed against the HIV-1 lifecycle. There are several reasons for why combination-based microbicides are being assessed in advanced preclinical and early clinical studies. 18,19 These drug combinations increase the broad spectrum of activity and can have additive or synergistic effects. Therefore, their concentrations can be decreased, resulting in potent activity against HIV-1 with fewer side-effects. These drug combinations also provide greater chances for HIV-1 protection, reducing the risk of resistance interfering at different stages in the HIV-1 lifecycle and acting against other sexually transmitted diseases (STDs) that fuel the HIV-1 epidemic, particularly genital herpes or herpes simplex virus type 2 (HSV-2). 20,21 In this context, tenofovir (TFV) and maraviroc (MRV) are the most extensively studied ARV, 22 -24 and anionic carbosilane dendrimers are novel, safe, and effective nanosystems directed against HIV-1 infection with great potential as topical microbicides. 25 -28 Some benefits of combining dendrimers with TFV and MRV potentially include a broad mechanism of action against nucleoside reverse transcrip-

tase inhibitor (NRTI)-resistant mutants, R5-HIV-1 and HIV-2 viruses and rare transmission events with X4-HIV virus without interfering in the antiviral effect of each ARV. Current approaches used in analysis of combined compound activities A variety of statistical methods for analysis have been widely used in studies involving the combined effects of two or more compounds. Chou and Talalay method is the most used in combinatorial studies. 29,30 It is based on the law of mass action. Drug interactions, the shape of their dose-effect curves and their half-maximal (50%)-effective concentration (EC50) were analyzed using CalcuSyn software. Briefly, this software takes into account the potency of each compound alone and in combination from dose–effect curves. To determine the EC50 of combinations, compounds are combined at an equipotent ratio at concentrations in excess of EC50 values, and serial dilutions of the combination are performed. Once the EC50 is determined, combination indexes (CIs) based on characterizing compounds as mutually exclusive, in other words, the compounds that have similar modes of action, were calculated as follows: CI ¼

ð f a =ð1− f a ÞÞ1 ð f a =ð1− f a ÞÞ2 þ ð f a =ð1− f a ÞÞC ð f a =ð1−f a ÞÞC

In this equation, fa is the fractional inhibition caused by a compound relative to a no-compound control, and the subscripts refer to the compound or combination used. CI values b 0.9 indicate a synergistic effect, 0.9 b CI b 1.1 indicate additive activity, and CI N 1.1 indicates antagonism. Tenofovir: clinical studies and efficacy as an HIV-1 microbicide TFV is an adenosine NRTI with potent activity against retroviruses, and it inhibits the synthesis of DNA by inserting into propagating viral DNA. 31 Therefore, TFV prevents initial cellular infection because it targets the HIV-1 lifecycle before genome integration. TFV gel (1%) was the first ARV showing the ability to prevent the sexual transmission of HIV-1. The results of the CAPRISA-004 clinical trial in a 1% TFV vaginal gel formulation provided the first evidence for preventing HIV and other STDs, such as HSV-2, in women; it reduced HIV acquisition by 39% overall and by 54% in women. 32 However, in the VOICE (MTN-003) clinical trial, TFV was ineffective when dosed once daily in a coitally dependent manner due to a lack of adherence. 33 The FACTS-001 clinical trial was designed to test the same regimen employed in CAPRISA-004 trial and seeks to assess its findings for possible product licensure and access. 34 The CAPRISA-008 trial is also an ongoing clinical trial designed to provide access to 1% TFV gel to HIV-negative CAPRISA-004 study participants to collect additional safety data and to implement a clinical care model through family planning services. 35 Other studies from the MTN-008 trial assessed the safety of 1% TFV gel in adolescents and menopausal, pregnant

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Figure 1. Schematic representation of dendrimer components. Core, end-groups, and the branching units. The most used nomenclature refers to when each generation corresponds to a layer of branching units.

and breastfeeding women and their interaction with other vaginal products. 36 Researchers from the MTN trials have carried out studies of 1% TFV gel as a rectal microbicide focusing on a high-risk population, which acquires HIV-1 through anal sex. The RMP-02/MTN-006 clinical trial, designed for rectal application once daily for 7 days was demonstrated not to be safe and acceptable for rectal use. 37 The MTN-007 clinical trial was designed to assess the safety, adherence, and acceptability of the reduced glycerin formulation of 1% TFV gel, and the results for the new formulation demonstrated that it was safe and well-tolerated rectally. 38 A subsequent study is currently ongoing in the MTN-017 phase II clinical trial, an expanded safety study of the 1% TFV gel used in the MTN-007 trial. If successful, results are expected in early 2015, setting the stage for a phase IIb/III trial. 39 The CHARM-01 and CHARM-02 trials compared the impact of three different TFV formulations on rectal safety and its acceptability, distribution and pharmacokinetics/pharmacodynamics profile. 40

Maraviroc: clinical studies and efficacy as an HIV-1 microbicide MRV (formerly UK-427,857) blocks CCR5-tropic HIV-1 entry into CD4 T-cells by binding to the CCR5 co-receptor. 41 MRV was selected through medical chemistry optimization of a high-throughput lead from a starting derivative taking into account parameters such as binding potency against the CCR5 co-receptor, antiviral activity, absorption, pharmacokinetics, and selectivity against key human targets. 41 The first clinical trial of a vaginal microbicide with MRV, separately and in combination was the MTN-013/IPM-026 trial. 42 The purpose of this study was to assess the safety and acceptability of the combination MRV/dapivarine (DPV, a non-nucleoside

reverse transcriptase inhibitor) vaginal ring shape when used continuously for 28 days. However, MRV did not absorb and work like DPV. 43 Because MRV is a promising candidate for development as a microbicide, the primary aim is to find its most effective combination. As the final goal, the CHARM Program suggests developing a vaginal/rectal microbicide that simultaneously has two or more compounds with different mechanisms of action, such as TFV/MRV. 39

Dendrimers: molecular structure, characterization, generations, and toxicity Dendrimers are highly branched nano-sized molecules with well-defined, homogeneous, and monodisperse structures consisting of tree-like arms or branches constructed through the sequential addition of branching units from an initiator. 44 -46 Molecular structure and components The size of most dendrimer-based nanomedicines ranges from 1 to 16 nm diameter and 30-200 kDa, which sits between the sizes of therapeutic proteins and liposomal or nanoparticulate delivery systems. 45,47 Compared with other nanocompounds, dendrimers are highly defined and have structural diversity. Dendrimer structures also differ from classical polymers 48 -50 and are divided into three main components: a central core, an interior shell surrounding the core, and a multivalent surface (Figure 1). The core affects the three-dimensional shape of the dendrimer, the interior affects the host-guest properties of the dendrimer, and the surface functional groups are the main factor for determining the ionic charge containing a large number of potentially reactive sites. 51 -53 The core and number of interior branching units also affect the dendrimer morphology. 53

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Figure 2. Chemical structures of commercially available dendrimers and commonly used as topical microbicides. (A) PAMAM, (B) poly(L-lysine), (C) Boltorn, (D) phosphorus-containing-dendrimer, (E) polypropylenimine, and (F) carbosilane-dendrimer. The structure of each dendrimer was drawn by using ChemSketch software.

The diameter of the dendrimers grows linearly according to added generations (at a rate of approximately 1 nm per generation), while the surface groups increase exponentially at each generation. 54,55 Characterization The most important aspect in the physicochemical characterization of dendrimers allows the development of consistent and interpretable results and inter-laboratory comparisons. The chemical structure, purity and functionality are characterized by nuclear magnetic resonance, mass spectrometric, and elemental analysis. 56 The presence of impurities, incomplete reaction, and reaction byproducts can be determined using matrixassisted laser desorption ionization time-of-light (MALDI-TOF) spectroscopy. 56 Other spectroscopic techniques are infra-red spectroscopy, Raman spectroscopy, fluorescence spectroscopy, X-ray photoelectron spectroscopy, electron paramagnetic resonance and X-ray scattering. 57 By using gel permeation chromatography (GPC), the absolute molecular weight and molecular size can be measured, and information on structure, conformation, aggregation and branching can be generated. 58 Scattering techniques allow to study their inter-molecular structure, intra-molecular cavity, radius-ofgyration, hydrodynamic radius, effective charge number, water penetration into the interior of the dendrimers, and the internal dynamics. 59 Structural composition can be also analyzed by atomic force microscopy, scanning thermal microscopy or electron microscopy. Potentiometric titrations and zeta potential analysis

provide crucial information on the net charge (the nature and integrity of dendrimer surfaces). 60 Chemical structures commonly used Several dendrimers have been synthesized and explored as topical microbicides against different STDs, and are shown in the Figure 2: polyamidoamine (PAMAM), poly(2,2-bis(hydroxymethyl)) propionic acid (Boltorn), polypropylenimine (PPI), poly(L-lysine) (PLL), phosphorus dendrimers (PPH) and carbosilane (C-Si) dendrimers. Synthesis Most dendrimers are synthesized following either a convergent or divergent route; the divergent approach proceeds from a multifunctional core outward, 46 and the convergent approach progresses from the surface groups inward to form a dendron that reacts with the multifunctional core (Figure 3). 61 Other technologies that have been employed for dendrimer synthesis include hypercore and branched monomers, double exponential, lego chemistry and click chemistry. 62 However, further studies are needed to select the most cost-effective synthesis strategies for their successful and correct commercialization. Polyanionic carbosilane dendrimers Most of reported carbosilane dendrimers have been synthesized via the divergent approach. From tetraallylsilane or tetravinylsilane as core, a hydrosilylation reaction with trichlorosilane and a nucleophilic displacement of chloride by allylmagnesium bromide complete the formation of the first generation. Subsequent generations are added by repetition of

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Figure 3. Approaches for the synthesis of dendrimers. (A) The divergent growth method, and (B) the convergent growth method.

the hydrosilylation and Grignard steps, and carbosilane dendrimers up to the fifth generation can be synthesized. 63 The strength of carbosilane dendrimers is related to the high energy of the C-Si bond and the low polarity, endowing it with high hydrophobicity. This can be modified by functionalization of the periphery with polar moieties, turning them hydrophilic. The presence of the 1,3,5-trihydroxybenzene core leads to carbosilane dendrimers less congested than related dendrimers with a silicon atom core. 64 Other synthetic strategies are based on the use of thiol-ene chemistry. Allyl-terminated carbosilane dendrimers are treated with thiols under UV-light to provide the functionalized dendrimers in short reaction times. The resulting solution is purified by nanofiltration through appropriate molecular weight cut-off membrane, and the solvent is eliminated by evaporation. 65 Synthetically and economically for use as microbicides against HIV/HSV-2, the use of carbosilane dendrimers at low generations (b G3) is more profitable than at high generations. The reaction time for the synthesis of the G3 allyl carbosilane dendrimers results too long (over 20 days) compared with G1 and G2 (2-6 days). 64 Moreover, from a biological perspective as antivirals, higher doses of dendrimers at low generations are used, and lower doses of dendrimers at high generations are needed to achieve the same antiviral efficacy. This is because we should take into account other associated factors such as cytotoxicity. Nomenclature Dendrimer sizes are classified by the generations, layers or repetitive units radically attached to the central core. The generation count does not follow a fixed rule and is not always consistent: the generation is determined by either taking into account that each generation corresponds to a layer of branching units or the number of repeating layers forming the dendrimer. 66

The first nomenclature is the most widely used to define PAMAM-type dendrimers. 67,68 Therefore, generation of dendrimers, from now on, will be referred to this first rule (Figure 1). Dendrimers at low generations have more flexible three dimensional shapes than dendrimers at high generations. The generation number of dendrimers (and the core) also reflects the rigidity of the overall structure. 46,53,69,70 Biocompatibility and toxicity The terms biocompatibility and toxicity when we discuss of dendrimers are very difficult to generalize because there are many factors to consider. Dendrimer cytotoxicity is relevant to the following features: surface charge, chemical composition, size and surface to volume ratio, and hydrophilicity/hydrophobicity of nano-architectures. 71,72 Surface characteristics have an important effect in efficacy, cellular uptake and toxicity of dendrimers. It is well-known that cationic dendrimers interact with negative biological membranes, enhancing the permeability and decreasing the integrity of membrane that finally causes its destabilization and cell lysis. 62,73 Moreover, various studies have shown that neutral and anionic dendrimers do not interact with biological membranes, consequently, are less toxic than cationic dendrimers and are mostly compatible for clinical application. 62,74 To discuss and to justify the use of anionic dendrimers in terms of toxicity and safety, we have to consider other factors such as, pharmacokinetics, biodistribution, route and frequency of administration. Polyanionic dendrimers can modulate cytokine/chemokine release, it is also important to define whether these dendrimers are biodegradable and what time they need to degradation because an incorrect use can produce unacceptable toxicity in clinical trials. Polyanionic carbosilane dendrimers Cytotoxicity in vitro has been evaluated using different cell lines and several assay methods. Variability in cell culture

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Table 1 Main characteristics of dendrimers against DC-SIGN as entry receptors for topical microbicide. Name (reference)

Dendrimer classification-generation

Central core (focal point)

Number and functional end groups

Stage of inhibition

EC50-values HIV-1 strains Cells

BH30sucMan 79

Boltorn-G3

2,2-bis(hydroxymethyl)-1, 3-propanediol

32 mannose units

EC50: 50 μM DC-SIGN-ECD

Dendron 12 (Polyman2)80

Boltorn-G3

3-Azidopropanoic acid

4 copies of linear pseudo-trimannoside

Inhibit gp120/DC-SIGN interaction Inhibit gp120/DC-SIGN interaction

Bol13.4 82

Boltorn-G1

Pentaerythritol

6 copies of linear pseudo-mannobioside with bis benzylamide

conditions makes it difficult to compare experiments conducted in different laboratories. Polyanionic carbosilane dendrimers (PCDs) are non-toxic at concentrations ranging from 20 to 100 μM in human epithelial cell lines derived from uterus (Hela MAGGI, TZM.bl, and HEC-1A), vagina (VK2/E6E7), and peripheral blood mononuclear cells (PBMCs). 75,76 Silicon-cored dendrimers are non-toxic up to 100 μM, while polyphenoxo-cored dendrimers show reduced biocompatibility at the different cell lines. 65 Lower generation carbosilane dendrimers show neither cytotoxicity nor significant changes on inflammatory cytokines profile, proliferation, microbiota or sperm survival at low concentrations of 10 μM. 64,65,75,76 Only a few studies on the in vivo toxicity of polyanionic dendrimers have been carried out so far. The general observation is that topical administration of dendrimers at G1 and G2 into CD1(ICR) mice and new Zealand white rabbits at low doses (1-50 μM) for 2 h and 24 h does not show vaginal irritation. 75,76 The administration of anionic dendrimers at G1 using BALB/c mice by topical use once daily for two consecutive days does not produce vaginal irritation and inflammation at concentrations up to 12 mM. Other studies assess the topical administration of dendrimers at G1 and G2 with TFV and MRV in BALB/c mice after daily treatment for 7 days. The results show that no histological lesions are presented at concentrations 400- to 4500-fold higher than anti-HIV-1 EC50-values in vitro. 28 Dendrimers as entry inhibitors for candidate topical microbicides The lack of efficacy of polyanionic polymers in the clinical trials and the increases in viral load 12 have cleared the way for novel non-specific HIV entry inhibitors as topical microbicides, such as dendrimers. Indeed, dendrimers are the only nanotechnology that has advances to human clinical trials as topical microbicide for HIV prophylaxis. Functionalized dendrimers as HIV entry inhibitors to prevent binding of HIV to the host cells can be classified into three groups according to the known mechanism of action and potential binding sites: Dendritic cell-specific intercellular adhesion molecule-3grabbing non-integrin (DC-SIGN) receptor, glycosphingolipids (GSLs) and the CD4/gp120 interaction.

Inhibit gp120/DC-SIGN interaction

EC50: 0.1 μM HIV-1BAL B-THP-1/DC-SIGN hCD4 +-T EC50: 5.9 μM HIV-1BAL B-THP-1/DC-SIGN hCD4 +-T

DC-SIGN receptors DC-SIGN are C-type lectins that recognize carbohydrates present on glycoproteins for several enveloped viruses, such as HIV. DC-SIGN capture viruses and transfer viral infection to other target cells enhancing viral entry and infection of cells that express the cognate entry receptor. 77 Glycodendrimers are dendrimers with carbohydrates at their surface useful as microbicides with high selectivity to interact with these specific lectin receptors. Structures based on second and third generation of Boltorn hyperbranched dendritic polymers functionalized with mannoses were evaluated for inhibition of HIV-1 entry with DC-SIGN as the target molecule. These structures, which are perfectly soluble in physiological conditions, non-toxic and easy to prepare, 78 inhibited the gp120/DC-SIGN interaction in the micromolar range. The best results were found for BH30sucMan with an EC50 of 50 μM (Table 1). 79 Tetravalent Boltorn-type dendrimers terminated with linear trimannoside mimics (dendron 12) reduced the trans-infection of CD4 T lymphocytes by over 90% at 50 μM, with almost complete inhibition at 100 μM. 80 Dendron 12 was also good tolerated by cervical explants and prevented HIV-1 infection by about 80% with an EC50 of 0.1 μM (Table 1). 81 A hexavalent presentation of Boltorn-type dendrimer terminated with linear pseudo-dimannoside groups and loaded with bisbenzylamide (Bol13.4) exhibited in a low micromolar range an HIV-1 inhibition of trans-infection of T cells in vitro (EC50: 5.9 μM) and provided 100% inhibition at 10 μM (Table 1). 82 This multivalent compound on a polyester backbone improved accessibility, and chemical stability to previously reported dendrimers. Nevertheless, these glycodendritic structures are recognized and degraded by mannosyl glycosylases. Thus, new strategies to address the stability of Boltorn-based glycodendrimers should be found to avoid mimic carbohydrates that are recognized by hydrolytic enzymes. 83 Glycosphingolipids as receptors GSLs are necessary for fusion of HIV-1 and host cell membranes in non-CD4 expressing cells. Galactose, cellobiose, globotriose and 3′-sialyllactose have been employed as

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Table 2 Main characteristics of dendrimers against several glycosphingolipids as entry receptors for topical microbicide. Name (reference)

Dendrimer classification-generation

Central core (focal point)

Number and functional end groups

Stage of inhibition

EC50-values HIV-1 strains Cells

PSGal64mer 85,86

PPI-G5

1,4-diaminobutane

gp120 attachment

BH3OPSGal 87

Boltorn-G3

2,2-bis(hydroxymethyl)-1, 3-propanediol

44 galactoses with 2 sulfate groups per galactose residue 32 β-galceramides

PPH-3d-G1 89

PPH-G1

Cyclotriphosphazene

12 galactosylceramides, N-hexadecylaminolactitol

gp120 attachment

PPH-5c-Gc′1 91

PPH-G1

Cyclotriphosphazene

12 phosponic acid moieties and lateral alkyl chains

gp120 attachment

SCSLD3 92

PLL-G3

Stearylamide

32 cellobioses. Degree of sulfation: 2.3

PLDG3-PSCel 93

PLL-G3

Benzhyldrylamine amide of L-lysine (divalent core)

24 cellobioses. Degree of sulfation: 1.9

Destroy HIV lipid bilayer by interaction with it gp120 attachment

TLACD3 95 ALACD3 95 MVC-GBT 97

PLL-G3 PLL-G3 PPI-G5

Tris(2-ethylamino)amine β-alanine methyl ester 1,4-diaminobutane

23 cellobioses 8 cellobioses 46 globotrioses

Non-determined yet Non-determined yet gp120 attachment

MVC-3SL 97

PPI-G5

1,4-diaminobutane

28 3′-sialyllactoses

gp120 attachment

Sulfo-6 98

PAMAM-G2

Ethylendiamine (EDA)

16 sialic acids with 11 sulfate groups

gp120 attachment

EC50: 0.6 nM HIV-1IIIB U373-MAGI-X4 EC50: 80 μM HIV-1BAL U373-MAGI-R5 EC50: 0.16-0.40 μM HIV-1LAI CEM-SS EC50: 1.0-1.5 μM HIV-1LAI CEM-SS and MT-4 EC50: 6.4 μg/ml HIV-1HTLV-IIIB MT-4 EC50: 3.2 μg/ml HIV-1HTLV-IIIB MT-4 Non-determined yet Non-determined yet EC50: 0.1-10.4 μg/ml HIV-1 primary strains H9, A3/R5 and PBMCs EC50: 0.2-15.6 μg/ml HIV-1 primary strains H9, A3/R5 and PBMCs EC50: 1.6-8.1 μM HIV-1 primary strains TZM.bl

functional dendrimer end groups to suppress the attachment of HIV to host cells. Galactosyl ceramide (GalCer) and its 3′-sulfated derivative are receptors that permit HIV-1 infection in CD4 − cells. 84 Different analogs of PPI tetrahexacontaamine GalCer-modified glycodendrimers, generations 1-5, showed efficiency against HIV-1. 85 Sulfated glycodendrimers were more potent than non-sulfated galactose-terminated dendrimers. Indeed, polysulfated galactose functionalized fifth generation (PSGal64mer) inhibited HIV-1 infectivity with EC50 values in the nanomolar range (EC50: 0.6-100 nM) (Table 2). 86 Two other Galcer-based dendrimers were reported for HIV-1 inhibition. One is based on two structures, third generation of Boltorn dendrimers with lower activity than PSGal64mer. BH3OPSGal showed an EC50 of 80 μM and had better behavior by the presence of sulfate groups in the periphery (Table 2). 87 Another group of GalCer glycodendrimers is based on PPH dendrimers. A collection of analogs based on first and second generation catanionic assemblies of phosphonic acid dendrimers and N-hexadecylamino-lactitol moieties was evaluated. 88 -90 All compounds exhibited a good antiviral activity (EC50: 0.16-0.40 μM), but they yielded low selectivity index, dramatically affected by 50% cytotoxicity concentration values (Table 2). 88,89 Thus, this series of dendrimers was deemed to be unsuitable for further development. The same group designed

gp120 attachment

other PPH dendrimers equipped with terminal phosphonic acid moieties and lateral alkyl chains. These dendrimers inhibited HIV-1 replication with EC50 values of 1-1.5 μM, with 5c-Gc′1 PPH-dendrimer as the most active (Table 2). 91 This study indicated the influence of pendant alkyl groups on the design and anti-HIV activity of dendrimers. Various types of sulfated cellobiose terminated PLL dendrimers have been evaluated to inhibit HIV entry. 92,93 A randomly sulfated cellobiose was connected to a generation 3 PLL dendrimer (PLDG3). Cellobiose alone does not have antiviral activity. 94 The biological activity has been ascribed to the cluster effects of randomly sulfated cellobiose, with an EC50 of 3.2 μg/ml (Table 2). 93 The same group evaluated the anti-HIV effect of third generation amphiphilic glycodendrimer synthesized from stearylamide lysine as core and sulfated cellobiose units as end groups of the dendrimer (SCSLD3). The main of this new synthesis lies on to immobilize the sulfated cellobiose cluster on a hydrophobic surface by hydrophobic interactions. The results showed that SCSLD3 had as high activity as PLDG3 (EC50: 6.7 μg/ml) (Table 2), indicating that the cluster effect and compact structure of cellobiose play an important role in the anti-HIV activity. 92 Spherical and hemispherical PLL dendrimers, generation 3, with cellobiose units (TLACD3 and ALACD3) were synthesized to evaluate the structural effects and the degree of sulfation on the anti-HIV activity (Table 2). 95

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Table 3 Main characteristics of dendrimers against gp120/CD4 interaction as entry step for topical microbicide. Name (reference) Dendrimer Central core classification-generation (focal point)

Number and functional end groups

Stage of inhibition

EC50-values HIV-1 strains Cells EC50: 0.01-2.2 μg/ml HIV-1 and SIVMAC MT-4, C8166 and PBMCs EC50: 0.1-3.5 μg/ml HIV-1 and SIVMAC MT-4, C8166 and PBMCs EC50: 0.73-2.35 μg/ml HIV-1 primary strains PBMCs EC50: 0.4-1.71 μg/ml HIV-1 primary strains PBMCs EC50: 0.72-1.60 μg/ml HIV-1 primary strains PBMCs EC50: 0.08-1.08 μM HIV-1 primary strains TZM.bl EC50: 13.1-36.5 nM HIV-1NL4.3, NLAD8 and HIV-2 TZM.bl, HEC-1A, VK2/E6E7 and PBMCs EC50: 15.9-50.7 nM HIV-1NL4.3, NLAD8 TZM.bl, HEC-1A, VK2/E6E7 and PBMCs EC50: 27.6 nM-1.1 μM HIV-1NL4.3, NLAD8 and 89.6 TZM.bl, HEC-1A, VK2/E6E7 and PBMCs EC50: 49.3 nM-1.2 μM HIV-1NL4.3, NLAD8 and 89.6 TZM.bl, HEC-1A, VK2/E6E7 and PBMCs EC50: 27.5-38.8 nM HIV-1NL4.3, NLAD8 and 89.6 TZM.bl

SPL2923 (BRI2923) 103

PAMAM-G4

Ammonia

24 1-(carboxymethoxy) naphthalene-3,6-disulfonate

SPL6195 (BRI6195) 103

PAMAM-G4

Ethylendiamine (EDA)

32 benzene dicarboxylate

gp120 attachment/fusion Retro-transcriptase Integration gp120 attachment/fusion

Benzhydrylamine amide of L-lysine (divalent core) Benzhyldrylamine amide of L-lysine (divalent core) Benzhydrylamine amide of L-lysine (divalent core) Benzhyldrylamine amide of L-lysine (divalent core) Silicon

32 1-(carboxymethoxy) naphthalene-3,6-disulfonate

gp120 attachment/fusion and replication

32 1-(carboxymethoxy) naphthalene-3,6-disulfonate

gp120 attachment/fusion Retro-transcriptase Integration gp120 attachment/fusion Retro-transcriptase Integration gp120 attachment/fusion

SPL7013 17,105,108 PLL-G4

SPL7304 105

PAMAM-G4

SPL7320 105

PPI-G4

SPL7115 17

PPL-G2

G1-S16 75,110

Carbosilane-G1

G2-STE16 and G2-CTE16 65

Carbosilane-G2 (thiol-ene synthesis)

Silicon

16 sulfonates and carboxylates CD4/gp120 interaction Virucide

G1-NS16 76,111

Carbosilane-G1

Silicon

16 naphthylsulphonates

CD4/gp120 interaction Virucide

G2-Sh1676,111

Carbosilane-G2

Silicon

16 sulphates

CD4/gp120 interaction Virucide

G2-S24P 64,110

Carbosilane-G2

Polyphenoxo

24 sulfonates

CD4/gp120 interaction Virucide

32 1-(carboxymethoxy) naphthalene-3,6-disulfonate 32 1-(carboxymethoxy) naphthalene-3,6-disulfonate 16 sulfonates

However, they are currently ongoing, and further revisions to study the relationship between antiviral and sulfated cellobiose should be taken into account. Globotriosyl ceramide and monosialoganglioside hematoside are major GSL constituents of B- and T-cell membranes. 96 It indicates that the interaction of HIV-1 with these GSLs involves certain surface determinants that are host cell-specific. Multivalent carbohydrates G5-PPI dendrimers terminated with globotriose (MVC-GBT) or 3′-sialyllactose (MVC-3SL) strongly inhibited HIV-1 infection by interactions between gp120 and cell surface with EC50 values ranging from 0.1 to N 100 μg/ml depending on type of sugar, cell lines and HIV-1 primary strains used (Table 2). 97 An initial trial set of different sulfated sialic acid terminated PAMAM glycodendrimers, generation 0-2, was also studied. 98 The generation 2 sulfated sialic acid-PAMAM glycodendrimer (sulfo-6) inhibited all HIV-1 strains with EC50 values in the low micromolar range (EC50: 1.6-8.1 μM)

CD4/gp120 interaction Virucide

(Table 2). However, this kind of dendrimer was much less potent as compared to dextran sulfate. CD4/gp120 docking One of the most promising targets of the HIV-1 cycle includes preventing the initial crucial steps in the infectious viral cycle: the viral entry/fusion process. The first step in virus entry is the attachment of viral gp120 (non-covalently linked to the transmembrane glycoprotein gp41) to the CD4 T-cell receptor. A conformational change in gp120 leads to binding to CCR5 and/or CXCR4 co-receptors on the cell. Finally, a conformational change in gp41 results in the fusion of the envelope with the cell membrane and the subsequent release of the viral capsid into the cytoplasm of the host cell. 99 Several polyanionic dendrimers have been evaluated and their anionic moieties provide a basis for the design of new systems

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Table 4 Main characteristics of dendrimers that require different receptors for cell entry as topical microbicide. Name (reference)

Dendrimer classification-generation

Central core (focal point)

Number and functional end groups

Stage of inhibition

EC50-values HIV-1 strains Cells

[G1]-CO2Na 114

GATG-G1

Gallic acid-triethylene glicol

9 benzoate groups

Inhibit dimerization of HIV-1 capsid protein (Assembly)

SB105-A10116

PLL-G2

Benzhyldrylamine amide of L-lysine (divalent core)

4 sequence peptide chains (ASLRVRIKK)

gp120/gp41 attachment

Viol36 117,118

Viologen-G1

Benzyl

6 ethyl units

CXCR4 antagonist

Viol7 117,118

Viologen-G1

Benzyl

6 thymine units

CXCR4 antagonist

Dissociation constant KD: 7.7 μM, detained at 25 °C by isothermal titration calorimetry EC50: 1.26-2.1 μg/ml HIV-1IIIB and HIV-1ada PBMCs EC50: 0.26-1.62 μg/ml HIV-1IIIB MT-4 and PBMCs EC50: 0.9 μg/ml HIV-1NL4.3 PBMCs

that inhibit viral fusion to cell membrane in a multivalent manner and overcome weak monovalent interactions to viral glycoproteins, such as gp120 and/or gp41. 17 There are regions in the HIV surface glycoprotein gp120 containing multiple basic amino acids that have been shown to interact with polyanions, the principle neutralizing domain (V3 loop), the C-terminal region, and conserved regions involved in chemokine coreceptor binding. 100-102 Several first to fifth generations of PAMAM dendrimers capped with different anionic surface groups were evaluated to establish structure-antiviral activity relationship. 17,103 All dendrimers inhibited gp120/CD4 complex at two levels: by weakness of the complex and by alteration of its dissociation pathway, potentially inhibiting HIV-1 entry. 104 Fourth generation phenyldicarboxylate (SPL6195) and naphthyldisulfonate (SPL2923)-terminated PAMAM dendrimers inhibited the replication of different HIV-1 strains with EC50 values of 0.1-3.5 μg/ml and 0.01-2.2 μg/ml, respectively (Table 3). Moreover, SPL2923 at 500-2500 times its EC50 was found to block the later stages of HIV infection, such as reverse transcription and integration processes.103 According to the structure and results of SPL2923, three dendrimers-generation 4 (PLL, PPI and PAMAM with ethylenediamine-core) with naphthyldisulfonate groups in the periphery were synthesized to enhance the HIV-1 antiviral activity. However, similar anti-HIV activity was found (EC50: 0.4-2.35 μg/ml; Table 3). 105 The level of residual cobalt and the risk of controlling on large-scale manufacture in PPI-based dendrimer (SPL7320), and the risk of reverse Michael reaction in PAMAM dendrimer in acidic formulation (SPL7304) do not make them as optimum candidates for research of new microbicides. Thus, PLL-dendrimer SPL7013 was elected and compared with another dendrimer of the same family (generation 2, SPL7115) 17 by its easy preparation on a large-scale, the optimum formulation compatibility, and stability. SPL7013 was more active than SPL7115 against HSV and blocked HIV-1 envelope mediated cell-to-cell fusion (Table 3). These results compared with other ARVs suggested that size (an elongated structure for SPL7115, or a compact structure for SPL7013), and multivalency of dendrimers are important for inhibiting cell-to-cell fusion.

The first topical nanomicrobicide to enter clinical trial is SPL7013, the active product in the candidate microbicide VivaGel™. 106 SPL7013 provided antiviral activity against HIV and HSV-2 in healthy women and men. 105 -107 However, the lack of broad anti-HIV activity against R5-HIV-1 strains 108 and the increased risk for HIV acquisition associated with epithelial injury after 7-14 days of twice-daily administration 109 are the main limitations of VivaGel™. New water-soluble PCDs, silicon-cored or polyphenoxocored 65,110,111 (Table 3) and other soluble copper carbosilane dendrimers 112 have been synthesized as topical microbicides. This issue will be addressed in the new section “Polyanionic carbosilane dendrimers as microbicides”. Others Due to the role of HIV-1 capsid protein during HIV-1 morphogenesis, novel potential inhibitors as microbicides based on disruption of capsid assembly and HIV-1 infectivity have emerged. 113 The main objective of these inhibitors is to destabilize the quaternary structure of C-terminal domain of the capsid protein and then the capsid protein assembly to form viral capsid. Several mannose, lactose, sulfonate and benzoateterminated gallic acid-triethylene glycol (GATG) dendrimers based on gallic acid core have been evaluated for this purpose. Only benzoate-terminated gallic acid dendrimer ([G1]-CO2Na) showed considerable inhibition of capsid protein dimerization and was able to hamper assembly of the HIV-1 capsid in vitro (Table 4). 114 Its incorporation at the focal point of GATG dendrimers confers them further solubility and biocompatibility, as well as better biodistribution. Heparan sulfate proteoglycans are cell membrane receptors composed of a core protein linked to sulfated glycosaminoglycans. 115 SB105-A10 is a peptide-derived dendrimer that contains a lysine core that tethers four peptide chains. It also exhibits a sequence of basic amino acids in the branching arms that can interact with viral glycoproteins and block virus attachment to proteoglycans by competitive inhibition. SB105-A10 inhibited HIV-1 infection with EC50 values ranging from 1.28 to 2.1 μg/ml (Table 4), inhibited the

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Figure 4. Schematic representation of several types of drug-dendrimer interactions. (A) Drug encapsulated dendrimer, (B) dendrimer-drug networks, (C) drug conjugated dendrimer by covalent interactions, and (D) by electrostatic interactions.

HIV infection in a biologically organotypic model of cervicovaginal epithelial tissue, and bound at a high affinity to the HIV envelope protein gp41 and, to lesser extent, gp120. 116 Several viologen derivatives (4,4-bipyridinium salts) dendrimers have been evaluated for their ability to inhibit HIV-1 replication by binding to HIV-1 surface proteins and/or on the host cell receptors required for entry. Most of the polycationic compounds showed good activities against HIV-1. In particular, comb-branched compounds Viol7 and Viol36 exhibited the highest activity (EC50: 0.9 μg/ml and 0.26-1.62 μg/ml, respectively), indicating that the antiviral activity requires an optimal number and distance of the positive charges (Table 4). 117,118 Moreover, they inhibited HIV-1 by interactions with the CXCR4 HIV co-receptor and were compared with the specific CXCR4 inhibitor plerixafor (AMD3100).

Polyanionic carbosilane dendrimers as microbicides New water-soluble PCDs, silicon-cored (G1-S16, G2-Sh16, G1-NS16, G2-STE16, G2-CTE16) or polyphenoxo-cored (G2-S24P), with sulfate, sulfonate or napthylsulfonate end groups have been synthesized 65,110,111 aiming at exhibiting more effective

and potent broad-spectrum anti-HIV and HSV-2 activity in vitro and in vivo and against other STDs 75,76 (Table 3). Molecular simulations confirmed that carbosilane dendrimers act as entry inhibitors, forming stable complexes with gp120, CD4 and, to a lesser extent, CCR5 and CXCR4 co-receptors. 75 These dendrimers provide a multifactorial and non-specific ability because they act as virucidal agents, inhibitors of entry, barriers to infection for long periods of time and inhibitors of cell-to-cell HIV-1 transmission. No irritation and vaginal lesions were detected in rabbits after 2 weeks of intravaginal application 75 or in female CD1(ICR) mice after dendrimers vaginal administration. 76 Topical vaginal dendrimer administration inhibited HIV-1 infection by 84% in humanized BLT (bone marrow-liver-thymus) mice without symptoms, including inflammation and vaginal irritation. Despite these encouraging results, several combination studies have the primary goal of achieving complete inhibition. Previous studies have shown the combination of different PCDs with ARVs (TFV and/or MRV) to act on different stages of the HIV-1 lifecycle. These combinations enhance the antiviral potency of individual compounds, even achieving 100% inhibition in different cell lines. 25 -28 As a result, anionic carbosilane dendrimers can be considered promising candidate compounds for topical microbicide application.

D. Sepúlveda-Crespo et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1481–1498 Table 5 Binding free energy for the dendrimer/CD4 and dendrimer/gp120 complexes by the MM-PBSA method. Dendrimer

G1-S16 G1-NS16 G2-Sh16 G2-STE16 G2-S24P

Binding free energy (ΔG) CD4

gp120 V3-root

gp120 (co-receptor binding site)

−15.8 −51.6 −68.3 − 42.5 − 39.2

− 38.0 − 86.2 − 94.3 − 90.0 − 9.6

− 41.7 − 107.0 − 120.6 − 111.4 Not-estimated

Molecular mechanics Poisson–Boltzmann surface area is used to estimate the binding free energy (ΔG) of biomolecular complexes. 137 More negative ΔG (kcal/mol) values indicate better binding (more favorable interaction). These values are compared to the thermodynamics of the CD4/gp120 binding interaction (ΔG = −9.5 kcal/mol)138 to verify possible competition and replacement by polyanionic carbosilane dendrimers.

Drug–dendrimer conjugated systems Several types of interactions of drugs with dendrimers have been proposed: entrapment of drugs within the internal cavities (involving encapsulation or dendrimer-drug networks) and interaction between the drug and the surface of the dendrimer (electrostatic or covalent interactions) (Figure 4). 62 Drug loading and entrapment efficiency is carried out by dialysis method. In this method, dendrimers in methanolic solution and aqueous solution of drug are mixed, following incubation for 24 h at room temperature. This solution is dialyzed twice in cellulose dialysis bag immersed in aqueous solution for 10 min to remove free drug from formulations, which is estimated spectrophotometrically to determine indirectly the amount of drug loaded within the system. Finally, the dialyzed formulations are lyophilized for further characterization. 119,120 The most studied anti-HIV drugs for inhibition of reverse transcription chemically conjugated to high generations of PAMAM or PPI dendrimers are lamivudine, efavirenz, and zidovudine. 121 -124 Drug–dendrimer interactions have great potential for drug delivery across barriers because of their small size and ease of surface functionalization of dendrimers to facilitate drug delivery. However, inherent toxicity associated with high generations of dendrimers has limited their application. Moreover, direct linkage of drugs might result in decreased therapeutic activity of the drug due to a change in its chemical structure and essentially requires drug release.

Combination of polyanionic carbosilane dendrimers with tenofovir PCDs block HIV-1 entry into cells, indicating that they bind to HIV-1 gp120 and/or the host cell receptors required for entry (Table 5). Combinations of TFV with a broad variety of PCDs, including G1-S16, G2-STE16 and G2-S24P (Table 6 and Figure 5, A-E) have recently been evaluated against X4-, R5- and X4/R5-tropic viruses in the TZM.bl cell line. Generations of dendrimers are defined according to the layers of branching units.

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The effects of the dual combination G2-STE16/TFV and the triple combinations G2-STE16/G2-S24P/TFV, G2-STE16/ G1-S16/TFV and G2-S24P/G1-S16/TFV 28 at a 2:2:1 fixed ratio against the R5-HIV-1NLAD8, X4-HIV-1NL4.3 and X4/R5-HIV-189.6 strains were assessed to determine which of the combinations was the strongest. The G2-STE16/G2-S24P/TFV combination has been shown to be the strongest because of the considerable decrease in the EC50 values for each of the compounds and the level of synergy as measured by CI values. By combining G2-STE16/G2-S24P/TFV, the EC50 for G2-STE16 and G2-S24P decreased against the three HIV-1 strains (EC50: 4.3-10.5 nM). A similar trend was observed for the TFV concentration (EC50: 2.6-4.7 nM). Treatments with low concentrations of 0.1 μM G2-STE16/0.1 μM G2-S24P/0.05 μM TFV resulted in 100% inhibition of X4-HIV-1NL4.3 and X4/R5-HIV-189.6, and 95% of R5-HIV-1NLAD8. CI calculations showed strong synergism at 75, 90, and 95% inhibition (CI: 0.08-0.30; Figure 6, A). These data suggest high-affinity binding of dendrimers to HIV-1 gp120, the CD4 cell receptor and CCR5/CXCR4 co-receptors. In particular, in the G2-STE16/G2-S24P/TFV combination, G2-STE16 is characterized by the greater affinity to HIV-1 gp120 and G2-S24P to the CD4 receptor and to smaller extent to CCR5 and/or CXCR4. G2-S24P provides additional support for viral entry when compared with the G2-STE16/TFV combination. Compared with the other two triple-combinations, G2-STE16 has a higher affinity for gp120 than G1-S16. It is also remarkable that the presence of G2-S24P in this group of combinations has a higher affinity for host-cell proteins because the combination is weakened by the absence of other ARVs that act at different stages of viral entry, as MRV. Furthermore, the G2-STE16/G2-S24P/TFV combination has several advantages related to the synthesis of G2-STE16 compared with dendrimers, which is performed via thiol-ene reaction: mild reaction conditions, high product yields with easy purification, and the ability to select the nature and density of functional groups by altering reactant ratios.

Combination of polyanionic carbosilane dendrimers with maraviroc In general, R5-viruses are the main strains present in body fluids (semen, blood, cervicovaginal and rectal secretions) and predominate in the early stages of HIV-1 infection. 125 -127 Combinations of G1-S16, G2-STE16 and G2-S24P with MRV have been evaluated against R5- and X4/R5-tropic HIV-1 strains. Our studies have shown that these dendrimers maintain their potent anti-HIV-1 activity in presence of ARV because MRV does not block X4-HIV-1NL4.3 infection or interfere with the antiviral activity of dendrimers. The effects of the dual combination G2-STE16/MRV 26 and the triple-combinations G2-STE16/G2-S24P/MRV, G2-STE16/ G1-S16/MRV and G2-S24P/G1-S16/MRV 28 at a 10:10:1 fixed ratio against R5-HIV-1NLAD8 and X4/R5-HIV-189.6 strains were evaluated to show which are the strongest. The three-dendrimer/ dendrimer/MRV combinations have similar EC50 values in the mixtures. Consequently, the percentage of inhibition against diverse HIV-1 strains and the level of synergy as measured by CI values demonstrated that G2-STE16/G1-S16/MRV is the strongest.

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Table 6 Chemical and structural characteristics of several commonly polyanionic carbosilane dendrimers used in combination with tenofovir and/or maraviroc for HIV-1 inhibition. Dendrimer

Molecular formula

MW (g/mol)

G

SG

NSG

Core

Ref.

G1-S16 G1-NS16 G2-Sh16 G2-STE16⁎ G2-S24P

C112H244N8Na16O48S16Si13 C184H244N24Na16O56S16Si13 C256H508N48Na16O64S16Si29 C144H300Na16O48S32Si13 C189H402N12Na24O75S24Si21

3717.2 4934.0 6978.4 4558.9 5954.4

1 1 2 2 2

Sulfonate Naphthyl-sulfonate Sulfate Sulfonate Sulfonate

16 16 16 16 24

Silicon Silicon Silicon Silicon Polyphenoxo

110 111 111 65 110

Abbreviations: G = Number of generations according to the number of branching units; MW = Molecular weight; NSG = Number of surface groups; SG = Surface groups. ⁎ Carbosilane dendrimer synthesized via thiol-ene chemistry.

Figure 5. Molecular representation of first- and second-generation dendrimers. (A) G1-S16, (B) G1-NS16, (C) G2-Sh16, (D) G2-STE16, and (E) G2-S24P. The generation of dendrimers is determined by considering that each generation corresponds to a layer of branching units.

By combining G2-STE16/G1-S16/MRV, a decrease in EC50 values for MRV against R5-HIV-1NLAD8 and X4/R5-HIV-189.6 (EC50 ~ 1.2 nM) infection was observed. The EC50 values for G2-STE16 and G1-S16 also decreased against both HIV-1 strains (EC50: 10.4-16.1 nM). Treatment with low concentrations of 5 μM G2-STE16/0.5 μM G1-S16/0.05 μM MRV resulted in 100% inhibition of R5-HIV-1NLAD8 and X4/R5-HIV-189.6. CI values also demonstrated potent synergistic interactions (CI: 0.07-0.24; Figure 6, B). By combining G2-STE16/G1-S16/MRV we obtained a microbicide that principally acts on the HIV-1 entry steps at different sites (envelope gp120, CD4 receptor, and CCR5/CXCR4 co-receptors), preventing the virus from binding these target cells. The ability to inhibit R5- and X4/R5-HIV-1 infection is due to the high affinity binding to gp120 by both dendrimers. G2-STE16 and G1-S16 are characterized by their high virucidal activity in a tropism-independent manner because they are able to reduce the

infectivity of viruses with contact for a short time. Therefore, the 100% inhibition observed may be due to total HIV-1 inactivation by the dendrimers, which enhance the mechanism when acting together without interfering with each other. Although the mechanism of action for virucidal activity by dendrimers remains unclear, it is likely an irreversible modification of the interaction domains of the HIV-1 gp120 protein with cellular receptors or capsid disintegration. Furthermore, if the virus is exposed to attack by the host cell, both dendrimers also have high-affinity blocking of the CD4 cell protein. The presence of MRV in this group of combinations enhances the protection process of viral entry. G2-STE16 and G1-S16 can also act against X4-viruses and strengthen their inhibitory activity together with MRV against R5-viruses. Moreover, we should not forget the advantages of synthesizing the G2-STE16 dendrimer.

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Figure 6. Combination indexes of different combinations at the EC95 level against HIV-1 infection in PBMCs or TZM.bl cells. The CI represents the mean ± SD calculated at the EC95 level from 1 to 3 independent combination experiments of polyanionic carbosilane dendrimers with (A) TFV, (B) MRV, or (C) TFV and MRV in TZM.bl cells or PBMC against R5-HIV-1NLAD8, X4-HIV-1NL4.3 or R5/X4-HIV-189.6.CI b 0.9 indicates a synergistic effect; 0.9 b CI b 1.1 indicates an additive effect (ad), and CI N 1.1 indicates antagonism.(1) The CI value corresponds to the EC50, the only concentration with synergistic interactions. A significant antagonistic effect at the EC75, EC90 and EC95 inhibitory concentrations was observed.(2) The CI value corresponds to the EC90 level, with strong synergistic interactions. From this value, a severe antagonistic effect was observed.

Combination of polyanionic carbosilane dendrimers with tenofovir and maraviroc Although R5-HIV-1 appears and predominates during early infection, X4-HIV-1 evolves at later stages, 126 and this should be taken into account when searching for an appropriate microbicide. Therefore, the major aim lies in studying the process involved in blocking HIV-1 infection acting on the early steps of viral replication before the integration processes. Dendrimers interact not only with viral gp120, but also with the CD4 and CCR5/CXCR4 receptors expressed on the cell surface, resulting in a potential multiple mechanism of action. In this manner,

combinations of TFV and MRV with PCDs including G1-S16, G2-STE16 and G2-S24P have been assessed against R5-, and X4/R5-tropic viruses in TZM.bl cells. The effects of the triple-combinations G2-STE16/TFV/MRV, G1-S16/TFV/MRV and G2-S24P/TFV/MRV 28 at a 10:5:1 fixed ratio against the R5-HIV-1NLAD8 and X4/R5-HIV-189.6 strains were evaluated to determine which of the combinations was the strongest. The three dendrimer/TFV/MRV combinations demonstrated low EC50 values in the mixtures. Consequently, the percentage of inhibition of diverse HIV-1 strains and the level of synergy measured as a CI value determined that G2-STE16/TFV/MRV is the strongest.

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By combining G2-STE16/TFV/MRV, a reduction in the EC50 for MRV against R5-HIV-1NLAD8 (EC50: 0.8 nM) or X4/R5-HIV-189.6 (EC50: 0.2 nM) infection was observed. The EC50 for G2-STE16 (EC50: 1.9-8.7 nM) and for TFV (EC50: 0.7-3.6 nM) also decreased. Greater than 95% inhibition of both HIV-1 strains with low concentrations of 0.1 μM G2-STE16/0.05 μM TFV/0.01 μM MRV was observed. CI displayed strongly synergistic interactions at the calculated EC75, EC90 and EC95 inhibitory concentrations (CI: 0.03-0.46; Figure 6, C). This group of combinations is characterized by the high virucidal activity of the dendrimers. The ability to inhibit R5- and X4/R5-HIV-1 infection is due to the high affinity binding to gp120 by G2-STE16, confirming the relevance of blocking and inactivating HIV-1 gp120. This fact is corroborated by taking into account that G1-S16/TFV/MRV and G2-S24P/TFV/MRV are the second and third strongest combinations, respectively. This result is because G1-S16 has a higher affinity for the virus than G2-S24P, but its affinity is less than G2-STE16. However, the presence of the additional support by MRV in hindering HIV-1 entry by blocking CCR5 is also remarkable. Finally, the presence of the G2-STE16 dendrimer is again noted in the strongest combination, having advantages for the above-mentioned synthesis.

strains (EC50 ~ 316 nM). Over 90% inhibition of R5-HIV-1NLAD8 and X4/R5-HIV-189.6 infection with 1 μM G1-NS16/1 μM MRV was observed. The CI values displayed synergism to additive effects (CI: ≤ 0.01-0.95; Figure 6, B). Data based on computer modeling related to the binding affinity of different proteins did not reach a convincing conclusion because G1-NS16 and G2-Sh16 dendrimers have high binding affinity values that are relatively similar. Therefore, we have to resort to other factors related to the mechanism of action of dendrimers in PBMCs due to the multifactorial and non-specific characteristics. There are no differences in the results found for the percentage of binding, inhibition of the gp120/CD4 interaction or the ability to inactivate the virus (corroborating the similar results obtained by computer modeling) by both dendrimers. However, there is a significant decrease in the expression of cell receptors and HIV-1 cell-cell transmission mostly from R5-tropic viruses in the presence of G1-NS16. Therefore, G1-NS16 bound to CD4 and CCR5 explains why G1-NS16 was slightly more efficient than G2-Sh16 at reducing the R5-HIV-1 transmission. G1-NS16 also acts against X4-viruses and strengthens their inhibitory activity with MRV against R5-viruses.

Combination of other classes of polyanionic carbosilane dendrimers

Clinical research: obstacles and challenges

It is becoming increasingly apparent that compounds directed against well-defined targets might have multiple mechanisms of antiviral action and against other viruses. Several generation of first and second carbosilane dendrimers, which also interfere with virus attachment by binding to HIV-1 gp120, inhibited HIV-1 infection of T-cell lines (PBMCs) using both laboratory-adapted and primary HIV-1 isolates. Combinations of several PCDs, including G1-NS16 and G2-Sh16 with TFV or MRV (Table 6 and Figure 5, A-E) have recently been evaluated against X4- and R5-tropic viruses in PBMCs. These combinations were also assessed against the human primary subtype C (R5)-HIV-1 strain, the most prevalent HIV-1 strain worldwide, and it may be the most transmissible strain compared with other subtypes. 128 Dendrimer generations are also defined by the layer of branching units. The strongest combinations are formed with G1-NS16 dendrimer. G1-NS16/TFV 25 and G1-NS16/MRV 27 are highly synergistic and stronger than combinations with G2-S16, which have low CI and EC50 values in the mixture. By combining G1-NS16/TFV, reductions in the EC50 values for G1-NS16 against R5-HIV-1NLAD8 (EC50: 0.03 nM) and X4-HIV-1NL4.3 (EC50: b 0.002 pM) infection were observed. The EC50 for TFV also decreased against both HIV-1 strains (EC50: 1.0 and b 0.005 pM, respectively). One hundred percent inhibition of both HIV-1 strains with low concentrations i.e., 0.1 μM G1-NS16/0.1 μM TFV and 0.05 μM G1-NS16/0.05 μM TFV, was observed. Treatment with 0.1 μM G1-NS16/0.1 μM TFV resulted in 85% inhibition against the subtype C-HIV-1 clinical strain. The CI values displayed strong synergism (CI ≤ 0.01; Figure 6, A). By combining G1-NS16/MRV, reductions in EC50 values for G1-NS16 against R5-HIV-1NLAD8 (EC50: 0.001 nM) and X4/R5-HIV-189.6 (EC50: 318 nM) infection were observed. The EC50 value for MRV also decreased against both HIV-1

In spite of the remarkable development with respect to the different dendrimers for microbicide applications and the success observed in in vitro studies, several polyanionic dendrimers have not moved beyond the preclinical stage. Some of the key obstacles include poor adherence, inability to overcome biological barriers, high toxicity by aggregation and accumulation within organs, unwarranted interactions with plasma proteins in systemic circulation, and the lack of adequate animal models for in vivo studies. Given the need for further microbicides and the failure of several nano-candidates in late stage clinical trials, one of the priorities is the development of an algorithm to assess microbicide safety and efficacy in a preclinical setting. This algorithm includes the evaluation of dendrimer efficacy and toxicity in relevant cell-based systems, the range and mechanism of action of candidate compounds, the interactions of the compound in combination with other approved ARVs, in vitro and in vivo pharmacokinetic, pharmacodynamics and safety evaluations and efficacy studies in sensitive models such as humanized mice. 129 Several obstacles have complicated the development of the most efficient combination-based microbicides. 130 The safety and efficacy of microbicides can only be designed with HIV infection as the primary endpoint. We should use adequate laboratory systems to avoid failures at the late stages of microbicide combination testing. We should also acquire more detailed knowledge for the evaluation and design of microbicides in clinical trials because we know little about the mechanism of HIV-1 transmission, the earliest stages of HIV-1 infection, and the role of host factors such as semen, 131 female genital tract secretions, 132 the physiology of mucosa due to the mixing of body fluids during sexual intercourse and mucosal responses against pathogens.

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Another main problem for microbicide combination development is the absence of a validated and standard animal model. Laboratory systems should include humanized mice to complement in vitro and in vivo findings to avoid the high cost and time of human studies. 133 Several logistical and ethical issues are involved in the use of combination-based microbicides. These matters include concerns about the safety of microbicide use during pregnancy and the exploitation of vulnerable populations to show the efficacy and safety of topical products. Another aspect to consider is that the threshold of acceptability, toxicity and efficacy of the product differs between countries. People should not fall into the risk compensation or behavioral disinhibition, which could reverse the beneficial effect of this product. Moreover, concerted efforts to engage men in microbicide use could make it easier for women to access and use microbicides in the future. 134 It is difficult to persuade pharmaceutical companies to support the research and development of microbicides overall (and more so in combination because it requires prior in-depth study of each of compounds individually). Nevertheless, funding for microbicide research has increased over the years, but not enough yet.

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approach for HIV prevention. A combination of several compounds that act preferentially on the early stages in the viral infection process, and inactivate the virus will likely ensure the most effective and powerful protection. Moreover, dually acting compounds targeting HIV co-pathogens (e.g., herpes viruses) should be taken into account to be included in combination microbicides. Combining different inhibitors that interfere with different stages in the transmission process and the viral cycle provides greater chances of protection and offers the possibility of synergy between different compounds, in which the activity of two or more compounds together is stronger than the sum of the two or more drugs alone. Combination based-microbicide vehicles are another important issue to consider for formulations, requiring that active compounds are stable in the vehicle and uniformly distributed throughout the luminal surface (vagina or rectum). 135 Vehicles can include topical gels, intravaginal rings, and locally applied solid films and tablets. 136 Nevertheless, novel routes of administration should be explored to maximize the activity, safety, acceptability and adherence of different nanotechnology-based prophylactic strategies for the prevention of HIV infection. Acknowledgments

Conclusions and outlook In the absence of an approved and effective HIV vaccine, the development of prevention strategies for controlling the HIV/AIDS epidemic is urgently needed. Several ARVs have moderate efficacy for preventing HIV transmission in human clinical trials. However, there are still associated problems, including toxicity, the emergence of multidrug-resistant mutants, systematic absorption, high daily cost, lifelong therapy, and possible drug–drug interactions. The use of single ARV microbicides has also decreased due to extreme polarity, poor adherence, and poor chemical or enzymatic stability. Continuing advances in the field of PrEP have provided new tools in the development of several types of nanosystems that contribute to the enhancement of current ARV therapy. These nanosystems have been shown to improve the permeability, solubility, stability and pharmacokinetics/pharmacodynamics properties of the prophylactic modalities. Moreover, they are also favorable for utilization in the field of microbicides due to their prolonged release of active compounds and their ability to penetrate epithelial mucosa. Knowing that the majority of HIV-affected individuals are from economically poor and undeveloped countries, several aspects need to be optimized for the successful translation of nanotechnology from the laboratory to clinical trials: biocompatibility, relatively low cost, the regulatory status of materials, reproducibility in in vitro and in vivo studies, ease of production and distribution, and potential for scale-up to large-scale manufacturing. Nanotechnology should also offer coitus-independent and long-term treatment to justify the cost associated with the use of this technology. These factors suggest the need for developing reliable microbicide strategies based on the combination of existing effective compounds for preventing HIV. Combination-based microbicides with diverse mechanisms of action may be the best

We thank Dr. Laura Diaz of the Flow Cytometry Unit (CA11/00290) for her technical assistance and Dr. Maria Isabel Clemente Mayoral for her technical assistance and advice regarding cell culture (CA10/01274).

References 1. Richman DD. HIV chemotherapy. Nature 2001;410:995-1001. 2. Harris M, Nosyk B, Harrigan R, Lima VD, Cohen C, Montaner J. Costeffectiveness of antiretroviral therapy for multidrug-resistant HIV: past, present, and future. AIDS Res Treat 2012;2012:595762. 3. Hajizadeh M, Sia D, Heymann SJ, Nandi A. Socioeconomic inequalities in HIV/AIDS prevalence in sub-Saharan African countries: evidence from the Demographic Health Surveys. Int J Equity Health 2014;13:18-40. 4. Maartens G, Celum C, Lewin SR. HIV infection: epidemiology, pathogenesis, treatment, and prevention. Lancet 2014;384:258-71. 5. Shen R, Richter HE, Smith PD. Interactions between HIV-1 and mucosal cells in the female reproductive tract. Am J Reprod Immunol 2014;71:608-17. 6. Murphy EM, Greene ME, Mihailovic A, Olupot-Olupot P. Was the “ABC” approach (abstinence, being faithful, using condoms) responsible for Uganda's decline in HIV? PLoS Med 2006;3:e379-84. 7. Gupta GR. How men's power over women fuels the HIV epidemic. BMJ 2002;324:183-4. 8. Smith PL, Tanner H, Dalgleish A. Developments in HIV-1 immunotherapy and therapeutic vaccination. F1000Prime Rep 2014;6:43-55. 9. Cottrell ML, Kashuba AD. Topical microbicides and HIV prevention in the female genital tract. J Clin Pharmacol 2014;54:603-15. 10. Peterson L, Nanda K, Opoku BK, Ampofo WK, Owusu-Amoako M, Boakye AY, et al. SAVVY (C31G) gel for prevention of HIV infection in women: a phase 3, double-blind, randomized, placebo-controlled trial in Ghana. PLoS One 2007;2:e1312-20. 11. Van Damme L, Ramjee G, Alary M, Vuylsteke B, Chandeying V, Rees H, et al. Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: a randomised controlled trial. Lancet 2002;360:971-7.

1496

D. Sepúlveda-Crespo et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1481–1498

12. Pirrone V, Wigdahl B, Krebs FC. The rise and fall of polyanionic inhibitors of the human immunodeficiency virus type 1. Antiviral Res 2011;90:168-82. 13. Ensign LM, Cone R, Hanes J. Nanoparticle-based drug delivery to the vagina: a review. J Control Release 2014;190C:500-14. 14. Kumar L, Verma S, Prasad DN, Bhardwaj A, Vaidya B, Jain AK. Nanotechnology: a magic bullet for HIV AIDS treatment. Artif Cells Nanomedicine Biotechnol 2014;43:71-86. 15. Gajbhiye V, Palanirajan VK, Tekade RK, Jain NK. Dendrimers as therapeutic agents: a systematic review. J Pharm Pharmacol 2009;61:989-1003. 16. Sanchez-Rodriguez J, Vacas-Cordoba E, Gomez R, De La Mata FJ, Munoz-Fernandez MA. Nanotech-derived topical microbicides for HIV prevention: the road to clinical development. Antivir Res 2015;113C:33-48. 17. Tyssen D, Henderson SA, Johnson A, Sterjovski J, Moore K, La J, et al. Structure activity relationship of dendrimer microbicides with dual action antiviral activity. PLoS One 2010;5:e12309-24. 18. D'Cruz OJ, Uckun FM. Vaginal microbicides and their delivery platforms. Expert Opin Drug Deliv 2014;11:723-40. 19. Kizima L, Rodriguez A, Kenney J, Derby N, Mizenina O, Menon R, et al. A potent combination microbicide that targets SHIV-RT, HSV-2 and HPV. PLoS One 2014;9:e94547-62. 20. Coates TJ, Richter L, Caceres C. Behavioural strategies to reduce HIV transmission: how to make them work better. Lancet 2008;372:669-84. 21. Freeman EE, Weiss HA, Glynn JR, Cross PL, Whitworth JA, Hayes RJ. Herpes simplex virus 2 infection increases HIV acquisition in men and women: systematic review and meta-analysis of longitudinal studies. AIDS 2006;20:73-83. 22. Chaowanachan T, Krogstad E, Ball C, Woodrow KA. Drug synergy of tenofovir and nanoparticle-based antiretrovirals for HIV prophylaxis. PLoS One 2013;8:e61416-29. 23. Cooper DA, Heera J, Ive P, Botes M, Dejesus E, Burnside R, et al. Efficacy and safety of maraviroc vs. efavirenz in treatment-naive patients with HIV-1: 5-year findings. AIDS 2014;28:717-25. 24. Ferir G, Palmer KE, Schols D. Synergistic activity profile of griffithsin in combination with tenofovir, maraviroc and enfuvirtide against HIV-1 clade C. Virology 2011;417:253-8. 25. Vacas-Cordoba E, Galan M, de la Mata FJ, Gomez R, Pion M, MunozFernandez MA. Enhanced activity of carbosilane dendrimers against HIV when combined with reverse transcriptase inhibitor drugs: searching for more potent microbicides. Int J Nanomedicine 2014;9:3591-600. 26. Sepulveda-Crespo D, Lorente R, Leal M, Gomez R, De la Mata FJ, Jimenez JL, et al. Synergistic activity profile of carbosilane dendrimer G2-STE16 in combination with other dendrimers and antiretrovirals as topical anti-HIV-1 microbicide. Nanomedicine 2014;10:609-18. 27. Cordoba EV, Arnaiz E, De La Mata FJ, Gomez R, Leal M, Pion M, et al. Synergistic activity of carbosilane dendrimers in combination with maraviroc against HIV in vitro. AIDS 2013;27:2053-8. 28. Sepulveda-Crespo D, Sánchez-Rodríguez J, Serramía MJ, Gomez R, De la Mata FJ, Jimenez JL, et al. Triple combination of carbosilane dendrimers, tenofovir and maraviroc as potential microbicide to prevent HIV-1 sexual transmission. Nanomedicine (Lond) 2015;10 [Epub ahead of print]. 29. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006;58:621-81. 30. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27-55. 31. De Clercq E. Clinical potential of the acyclic nucleoside phosphonates cidofovir, adefovir, and tenofovir in treatment of DNA virus and retrovirus infections. Clin Microbiol Rev 2003;16:569-96. 32. Sokal DC, Karim QA, Sibeko S, Yende-Zuma N, Mansoor LE, Baxter C, et al. Safety of tenofovir gel, a vaginal microbicide, in South African

33.

34. 35.

36.

37.

38.

39. 40.

41.

42.

43. 44.

45.

46.

47.

48.

49. 50. 51.

52. 53. 54.

women: results of the CAPRISA 004 Trial. Antivir Ther 2013;18:301-10. Celum C, Baeten JM. Tenofovir-based pre-exposure prophylaxis for HIV prevention: evolving evidence. Curr Opin Infect Dis 2012;25:51-7. FACTS. FACTS 001 Study Design. In, http://www.facts-consortium. co.za/. Clinical Trials. Implementation effectiveness and safety of tenofovir gel provision through family planning services. In, http://clinicaltrials.gov/ show/NCT01691768. MTN. MTN-008 Expanded Safety Study of Tenofovir Gel in Pregnant and Breastfeeding Women. In, http://www.mtnstopshiv. org/news/studies/mtn008. Anton PA, Cranston RD, Kashuba A, Hendrix CW, Bumpus NN, Richardson-Harman N, et al. RMP-02/MTN-006: a phase 1 rectal safety, acceptability, pharmacokinetic, and pharmacodynamic study of tenofovir 1% gel compared with oral tenofovir disoproxil fumarate. AIDS Res Hum Retroviruses 2012;28:1412-21. McGowan I, Hoesley C, Cranston RD, Andrew P, Janocko L, Dai JY, et al. A phase 1 randomized, double blind, placebo controlled rectal safety and acceptability study of tenofovir 1% gel (MTN-007). PLoS One 2013;8:e6014-23. McGowan I. The development of rectal microbicides for HIV prevention. Expert Opin Drug Deliv 2014;11:69-82. MTN. MTN-013/IPM 026: Phase I Safety and Drug Absorption Trial of a Combination Dapivirine-Maraviroc Vaginal Ring. In, http://www. mtnstopshiv.org/news/studies/mtn013. Dorr P, Westby M, Dobbs S, Griffin P, Irvine B, Macartney M, et al. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broadspectrum anti-human immunodeficiency virus type 1 activity. Antimicrob Agents Chemother 2005;49:4721-32. Malcolm RK, Fetherston SM, McCoy CF, Boyd P, Major I. Vaginal rings for delivery of HIV microbicides. Int J Womens Health 2012;4:595-605. MTN. Rectal Microbicides Fact Sheet. In, http://www.mtnstopshiv. org/node/2864. Tomalia DA. Birth of a new macromolecular architecture: dendrimers as quantized building blocks for nanoscale synthetic polymer chemistry. Prog Polym Sci 2005;30:294-324. Tomalia DA. Dendrons/dendrimers: quantized, nano-element like building blocks for soft-soft and soft-hard nano-compound synthesis. Soft Matter 2010;6:456-74. Tomalia DA, Baker H, Dewald J, Hall M, Kallos S, Roeck J, et al. A new class of polymers: starburst-dendritic macromolecules. Polym J 1985;17:117-32. Kaminskas LM, Boyd BJ, Porter CJ. Dendrimer pharmacokinetics: the effect of size, structure and surface characteristics on ADME properties. Nanomedicine (Lond) 2011;6:1063-84. Abbasi E, Aval SF, Akbarzadeh A, Milani M, Nasrabadi HT, Joo SW, et al. Dendrimers: synthesis, applications, and properties. Nanoscale Res Lett 2014;9:247-57. Ina M. Dendrimer: a novel drug delivery system. J Drug Deliv Ther 2011;1:70-4. Tripathy S, Das MK. Dendrimers and their applications as novel drug delivery carriers. J Appl Pharm Sci 2013;3:142-9. Kannan RM, Nance E, Kannan S, Tomalia DA. Emerging concepts in dendrimer-based nanomedicine: from design principles to clinical applications. J Intern Med 2014;276:579-617. Lombardo D. Modeling dendrimers charge interaction in solution: relevance in biosystems. Biochem Res Int 2014;2014:837651. Mintzer MA, Grinstaff MW. Biomedical applications of dendrimers: a tutorial. Chem Soc Rev 2011;40:173-90. Tomalia DA. Birth of a new macromolecular architecture: dendrimers as quantized building blocks for nanoscale synthetic organic chemistry. Aldrichimica Acta 2004;37:39-57.

D. Sepúlveda-Crespo et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1481–1498 55. Svenson S, Tomalia DA. Dendrimers in biomedical applications— reflections on the field. Adv Drug Deliv Rev 2005;57:2106-29. 56. Ma CQ, Mena-Osteritz E, Wunderlin M, Schulz G, Bauerle P. 2,2′:3′,2″-Terthiophene-based all-thiophene dendrons and dendrimers: synthesis, structural characterization, and properties. Chemistry 2012;18:12880-901. 57. Gautam SP, Gupta AK, Agrawal S, Sureka S. Spectroscopic characterization of dendrimers. Int J Pharm Pharm Sci 2012;4:77-80. 58. Hedden RC, Bauer BJ. Structure and dimensions of PAMAM/PEG dendrimer–star polymers. Macromolecules 2003;36:1829-35. 59. Wang X, Guerrand L, Wu B, Boldon L, Chen W-R, Liu L. Characterizations of polyamidoamine dendrimers with scattering techniques. Polymers 2012;4:600-16. 60. Li J, Piehler LT, Qin D, Baker Jr JR, Tomalia DA. Visualization and characterization of poly(amidoamine) dendrimers by atomic force microscopy. Langmuir 2000;16:5613-6. 61. Hawker CJ, Fréchet JMJ. Preparation of polymers with controlled molecular architecture. A new convergent approach to dendritic macromolecules. J Am Chem Soc 1990;112:7638-47. 62. Madaan K, Kumar S, Poonia N, Lather V, Pandita D. Dendrimers in drug delivery and targeting: drug-dendrimer interactions and toxicity issues. J Pharm Bioallied Sci 2014;6:139-50. 63. Son DY. A durable template for carbosilane dendrimer synthesis. Chem Commun (Camb) 2013;49:10209-10. 64. Sánchez-Nieves J, Ortega P, Muñoz-Fernandez MA, Gomez R, De la Mata FJ. Synthesis of carbosilane dendrons and dendrimers derived from 1,3,5-trihydroxybenzene. Tetrahedron 2010;66:9203-13. 65. Galan M, Sanchez Rodriguez J, Jimenez JL, Relloso M, Maly M, de la Mata FJ, et al. Synthesis of new anionic carbosilane dendrimers via thiol-ene chemistry and their antiviral behaviour. Org Biomol Chem 2014;12:3222-37. 66. Dufes C, Uchegbu IF, Schatzlein AG. Dendrimers in gene delivery. Adv Drug Deliv Rev 2005;57:2177-202. 67. Esfand R, Tomalia DA. Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discov Today 2001;6:427-36. 68. Kaczorowska MA, Cooper HJ. Electron capture dissociation, electron detachment dissociation, and collision-induced dissociation of polyamidoamine (PAMAM) dendrimer ions with amino, amidoethanol, and sodium carboxylate surface groups. J Am Soc Mass Spectrom 2008;19:1312-9. 69. Bosman AW, Janssen HM, Meijer EW. About dendrimers: structure, physical properties, and applications. Chem Rev 1999;99:1665-88. 70. de Gennes PG, Hervet H. Statistics of “starburst” polymers. J Phys Lett 1983;44:351-60. 71. Duncan R, Izzo L. Dendrimer biocompatibility and toxicity. Adv Drug Deliv Rev 2005;57:2215-37. 72. Greish K, Thiagarajan G, Herd H, Price R, Bauer H, Hubbard D, et al. Size and surface charge significantly influence the toxicity of silica and dendritic nanoparticles. Nanotoxicology 2012;6:713-23. 73. Domanski DM, Klajnert B, Bryszewska M. Influence of PAMAM dendrimers on human red blood cells. Bioelectrochemistry 2004;63:189-91. 74. Jevprasesphant R, Penny J, Jalal R, Attwood D, McKeown NB, D'Emanuele A. The influence of surface modification on the cytotoxicity of PAMAM dendrimers. Int J Pharm 2003;252:263-6. 75. Chonco L, Pion M, Vacas E, Rasines B, Maly M, Serramia MJ, et al. Carbosilane dendrimer nanotechnology outlines of the broad HIV blocker profile. J Control Release 2012;161:949-58. 76. Vacas Cordoba E, Arnaiz E, Relloso M, Sanchez-Torres C, Garcia F, Perez-Alvarez L, et al. Development of sulphated and naphthylsulphonated carbosilane dendrimers as topical microbicides to prevent HIV-1 sexual transmission. AIDS 2013;27:1219-29. 77. Lozach PY, Burleigh L, Staropoli I, Amara A. The C type lectins DCSIGN and L-SIGN: receptors for viral glycoproteins. Methods Mol Biol 2007;379:51-68.

1497

78. Arce E, Nieto PM, Diaz V, Castro RG, Bernad A, Rojo J. Glycodendritic structures based on Boltorn hyperbranched polymers and their interactions with Lens culinaris lectin. Bioconjug Chem 2003;14:817-23. 79. Tabarani G, Reina JJ, Ebel C, Vives C, Lortat-Jacob H, Rojo J, et al. Mannose hyperbranched dendritic polymers interact with clustered organization of DC-SIGN and inhibit gp120 binding. FEBS Lett 2006;580:2402-8. 80. Sattin S, Daghetti A, Thepaut M, Berzi A, Sanchez-Navarro M, Tabarani G, et al. Inhibition of DC-SIGN-mediated HIV infection by a linear trimannoside mimic in a tetravalent presentation. ACS Chem Biol 2010;5:301-12. 81. Berzi A, Reina JJ, Ottria R, Sutkeviciute I, Antonazzo P, SanchezNavarro M, et al. A glycomimetic compound inhibits DC-SIGNmediated HIV infection in cellular and cervical explant models. AIDS 2012;26:127-37. 82. Varga N, Sutkeviciute I, Ribeiro-Viana R, Berzi A, Ramdasi R, Daghetti A, et al. A multivalent inhibitor of the DC-SIGN dependent uptake of HIV-1 and dengue virus. Biomaterials 2014;35:4175-84. 83. Rojo J, Delgado R. Glycodendritic structures: promising new antiviral drugs. J Antimicrob Chemother 2004;54:579-81. 84. Harouse JM, Collman RG, Gonzalez-Scarano F. Human immunodeficiency virus type 1 infection of SK-N-MC cells: domains of gp120 involved in entry into a CD4-negative, galactosyl ceramide/3' sulfogalactosyl ceramide-positive cell line. J Virol 1995;69:7383-90. 85. Kensinger RD, Yowler BC, Benesi AJ, Schengrund CL. Synthesis of novel, multivalent glycodendrimers as ligands for HIV-1 gp120. Bioconjug Chem 2004;15:349-58. 86. Kensinger RD, Catalone BJ, Krebs FC, Wigdahl B, Schengrund CL. Novel polysulfated galactose-derivatized dendrimers as binding antagonists of human immunodeficiency virus type 1 infection. Antimicrob Agents Chemother 2004;48:1614-23. 87. Morales-Serna JA, Boutureira O, Serra A, Matheu MI, Diaz Y, Castillon S. Synthesis of hyperbranched β-galceramide-containing dendritic polymers that bind HIV-1 rgp120. Eur J Org Chem 2010;2010:2657-60. 88. Blanzat M, Turrin CO, Aubertin AM, Couturier-Vidal C, Caminade AM, Majoral JP, et al. Dendritic catanionic assemblies: in vitro antiHIV activity of phosphorus-containing dendrimers bearing galbeta1cer analogues. Chembiochem 2005;6:2207-13. 89. Perez-Anes A, Stefaniu C, Moog C, Majoral JP, Blanzat M, Turrin CO, et al. Multivalent catanionic GalCer analogs derived from first generation dendrimeric phosphonic acids. Bioorg Med Chem 2010;18:242-8. 90. Blanzat M, Turrin CO, Perez E, Rico-Lattes I, Caminade AM, Majoral JP. Phosphorus-containing dendrimers bearing galactosylceramide analogs: self-assembly properties. Chem Commun (Camb) 2002:1864-5. 91. Perez-Anes A, Spataro G, Coppel Y, Moog C, Blanzat M, Turrin CO, et al. Phosphonate terminated PPH dendrimers: influence of pendant alkyl chains on the in vitro anti-HIV-1 properties. Org Biomol Chem 2009;7:3491-8. 92. Han S, Kanamoto T, Nakashima H, Yoshida T. Synthesis of a new amphiphilic glycodendrimer with antiviral functionality. Carbohydr Polym 2012;90:1061-8. 93. Han S, Yoshida D, Kanamoto T, Nakashima H, Uryu T, Yoshida T. Sulfated oligosaccharide cluster with polylysine core scaffold as a new anti-HIV dendrimer. Carbohydr Polym 2010;80:1111-5. 94. Lundquist JJ, Toone EJ. The cluster glycoside effect. Chem Rev 2002;102:555-78. 95. Han S, Baigude H, Yoshida T, Uryu T. Synthesis of new spherical and hemispherical oligosaccharides with polylysine core scaffold. Carbohydr Polym 2007;68:26-34. 96. Degroote S, Wolthoorn J, van Meer G. The cell biology of glycosphingolipids. Semin Cell Dev Biol 2004;15:375-87. 97. Rosa Borges A, Wieczorek L, Johnson B, Benesi AJ, Brown BK, Kensinger RD, et al. Multivalent dendrimeric compounds containing

1498

98.

99. 100.

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

113. 114.

115. 116.

D. Sepúlveda-Crespo et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1481–1498 carbohydrates expressed on immune cells inhibit infection by primary isolates of HIV-1. Virology 2010;408:80-8. Clayton R, Hardman J, LaBranche CC, McReynolds KD. Evaluation of the synthesis of sialic acid-PAMAM glycodendrimers without the use of sugar protecting groups, and the anti-HIV-1 properties of these compounds. Bioconjug Chem 2011;22:2186-97. Pierson TC, Doms RW. HIV-1 entry and its inhibition. Curr Top Microbiol Immunol 2003;281:1-27. Huang CC, Tang M, Zhang MY, Majeed S, Montabana E, Stanfield RL, et al. Structure of a V3-containing HIV-1 gp120 core. Science 2005;310:1025-8. Meshcheryakova D, Andreev S, Tarasova S, Sidorova M, Vafina M, Kornilaeva G, et al. CD4-derived peptide and sulfated polysaccharides have similar mechanisms of anti-HIV activity based on electrostatic interactions with positively charged gp120 fragments. Mol Immunol 1993;30:993-1001. Rizzuto CD, Wyatt R, Hernandez-Ramos N, Sun Y, Kwong PD, Hendrickson WA, et al. A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding. Science 1998;280:1949-53. Witvrouw M, Fikkert V, Pluymers W, Matthews B, Mardel K, Schols D, et al. Polyanionic (i.e., polysulfonate) dendrimers can inhibit the replication of human immunodeficiency virus by interfering with both virus adsorption and later steps (reverse transcriptase/integrase) in the virus replicative cycle. Mol Pharmacol 2000;58:1100-8. Nandy B, Bindu DH, Dixit NM, Maiti PK. Simulations reveal that the HIV-1 gp120-CD4 complex dissociates via complex pathways and is a potential target of the polyamidoamine (PAMAM) dendrimer. J Chem Phys 2013;139:024905-9. McCarthy TD, Karellas P, Henderson SA, Giannis M, O'Keefe DF, Heery G, et al. Dendrimers as drugs: discovery and preclinical and clinical development of dendrimer-based microbicides for HIV and STI prevention. Mol Pharm 2005;2:312-8. Rupp R, Rosenthal SL, Stanberry LR. VivaGel (SPL7013 Gel): a candidate dendrimer–microbicide for the prevention of HIV and HSV infection. Int J Nanomedicine 2007;2:561-6. Price CF, Tyssen D, Sonza S, Davie A, Evans S, Lewis GR, et al. SPL7013 Gel (VivaGel(R)) retains potent HIV-1 and HSV-2 inhibitory activity following vaginal administration in humans. PLoS One 2011;6:e24095-107. Telwatte S, Moore K, Johnson A, Tyssen D, Sterjovski J, Aldunate M, et al. Virucidal activity of the dendrimer microbicide SPL7013 against HIV-1. Antiviral Res 2011;90:195-9. Moscicki AB, Kaul R, Ma Y, Scott ME, Daud II, Bukusi EA, et al. Measurement of mucosal biomarkers in a phase 1 trial of intravaginal 3% StarPharma LTD 7013 gel (VivaGel) to assess expanded safety. J Acquir Immune Defic Syndr 2012;59:134-40. Rasines B, Sanchez-Nieves J, Maiolo M, Maly M, Chonco L, Jimenez JL, et al. Synthesis, structure and molecular modelling of anionic carbosilane dendrimers. Dalton Trans 2012;41:12733-48. Arnáiz E, Vacas Cordoba E, Galan M, Pion M, Gomez R, MunozFernandez MA, et al. Synthesis of anionic carbosilane dendrimers via “click chemistry” and their antiviral properties against HIV. J Polym Sci A1 2014;52:1099-112. Galan M, Sanchez-Rodriguez J, Cangiotti M, Garcia-Gallego S, Jimenez JL, Gomez R, et al. Antiviral properties against HIV of water soluble copper carbosilane dendrimers and their EPR characterization. Curr Med Chem 2012;19:4984-94. Neira JL. The capsid protein of human immunodeficiency virus: designing inhibitors of capsid assembly. FEBS J 2009;276:6110-7. Domenech R, Abian O, Bocanegra R, Correa J, Sousa-Herves A, Riguera R, et al. Dendrimers as potential inhibitors of the dimerization of the capsid protein of HIV-1. Biomacromolecules 2010;11:2069-78. Bishop JR, Schuksz M, Esko JD. Heparan sulphate proteoglycans finetune mammalian physiology. Nature 2007;446:1030-7. Bon I, Lembo D, Rusnati M, Clo A, Morini S, Miserocchi A, et al. Peptide-derivatized SB105-A10 dendrimer inhibits the infectivity of R5

117. 118.

119.

120.

121.

122.

123.

124. 125.

126. 127.

128.

129.

130.

131.

132.

133. 134.

135.

136. 137. 138.

and X4 HIV-1 strains in primary PBMCs and cervicovaginal histocultures. PLoS One 2013;8:e76482-95. Asaftei S, De Clercq E. "Viologen" dendrimers as antiviral agents: the effect of charge number and distance. J Med Chem 2010;53:3480-8. Asaftei S, Huskens D, Schols D. HIV-1 X4 activities of polycationic “viologen” based dendrimers by interaction with the chemokine receptor CXCR4: study of structure-activity relationship. J Med Chem 2012;55:10405-13. Kumar PD, Kumar PV, Selvam TP, Rao KS. PEG conjugated PAMAM dendrimers with a anti-HIV drug stavudine for prolong release. Res Biotechnol 2013;4:10-8. Kumar PD, Kumar PV, Selvam TP, Rao KS. Prolonged drug delivery system of PEGylated PAMAM dendrimers with a anti-HIV drug. Res Pharm 2013;3:08-17. Dutta T, Garg M, Jain NK. Targeting of efavirenz loaded tuftsin conjugated poly(propyleneimine) dendrimers to HIV infected macrophages in vitro. Eur J Pharm Sci 2008;34:181-9. Dutta T, Jain NK. Targeting potential and anti-HIV activity of lamivudine loaded mannosylated poly (propyleneimine) dendrimer. Biochim Biophys Acta 2007;1770:681-6. Gajbhiye V, Ganesh N, Barve J, Jain NK. Synthesis, characterization and targeting potential of zidovudine loaded sialic acid conjugated-mannosylated poly(propyleneimine) dendrimers. Eur J Pharm Sci 2013;48:668-79. Pyreddy S, Kumar PD, Kumar PV. Polyethylene glycolated PAMAM dendrimers-Efavirenz conjugates. Int J Pharm Invest 2014;4:15-8. Balandya E, Sheth S, Sanders K, Wieland-Alter W, Lahey T. Semen protects CD4+ target cells from HIV infection but promotes the preferential transmission of R5 tropic HIV. J Immunol 2010;185:7596-604. Grivel JC, Shattock RJ, Margolis LB. Selective transmission of R5 HIV-1 variants: where is the gatekeeper? J Transl Med 2011;9(Suppl 1):S1-6. Sarrami-Forooshani R, Mesman AW, van Teijlingen NH, Sprokholt JK, van der Vlist M, Ribeiro CM, et al. Human immature Langerhans cells restrict CXCR4-using HIV-1 transmission. Retrovirology 2014;11:52. Kahle E, Campbell M, Lingappa J, Donnell D, Celum C, Ondondo R, et al. HIV-1 subtype C is not associated with higher risk of heterosexual HIV-1 transmission: a multinational study among HIV-1 serodiscordant couples. AIDS 2014;28:235-43. Sanchez-Rodriguez J, Vacas-Cordoba E, Gomez R, De La Mata FJ, MunozFernandez MA. Nanotech-derived topical microbicides for HIV prevention: the road to clinical development. Antiviral Res 2015;113C:33-48. Abdool Karim SS, Baxter C. Overview of microbicides for the prevention of human immunodeficiency virus. Best Pract Res Clin Obstet Gynaecol 2012;26:427-39. Zirafi O, Kim K, Roan NR, Kluge SF, Müller JA, Jiang S, et al. Semen enhances HIV infectivity and impairs the antiviral efficacy of microbicides. Sci Transl Med 2014;6:262-73. Herold BC, Mesquita PM, Madan RP, Keller MJ. Female genital tract secretions and semen impact the development of microbicides for the prevention of HIV and other sexually transmitted infections. Am J Reprod Immunol 2011;65:325-33. Denton PW, Garcia JV. Humanized mouse models of HIV infection. AIDS Rev 2011;13:135-48. Lanham M, Wilcher R, Montgomery ET, Pool R, Schuler S, Lenzi R, et al. Engaging male partners in women's microbicide use: evidence from clinical trials and implications for future research and microbicide introduction. J Int AIDS Soc 2014;17:19159-70. Nunes R, Sarmento B, das Neves J. Formulation and delivery of antiHIV rectal microbicides: advances and challenges. J Control Release 2014;194C:278-94. Rohan LC, Devlin B, Yang H. Microbicide dosage forms. Curr Top Microbiol Immunol 2014;383:27-54. Kuhn B, Gerber P, Schulz-Gasch T, Stahl M. Validation and use of the MM-PBSA approach for drug discovery. J Med Chem 2005;48:4040-8. Myszka DG, Sweet RW, Hensley P, Brigham-Burke M, Kwong PD, Hendrickson WA, et al. Energetics of the HIV gp120-CD4 binding reaction. Proc Natl Acad Sci U S A 2000;97:9026-31.