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PAMAM Dendrimers: Destined for Success or Doomed to Fail? Plain and Modified PAMAM Dendrimers in the Context of Biomedical Applications MAGDALENA LABIENIEC-WATALA,1 CEZARY WATALA2 1 2

University of Lodz, Faculty of Biology and Environmental Protection, Department of Thermobiology, Lodz 90-236, Poland Department of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences, Medical University of Lodz, Lodz 92-215, Poland

Received 29 June 2014; revised 2 October 2014; accepted 3 October 2014 Published online 31 October 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24222 ABSTRACT: PAMAM (polyamidoamine) dendrimers are commonly considered promising polymers that can be successfully used in various biomedical applications. Nevertheless, direct clinical adaptations of plain unmodified PAMAM dendrimers may be limited at present, mainly because of their toxicity, unpredictable behavior in living organisms, unknown bioavailability, biocompatibility or pharmacokinetic profile, problematic therapeutic dose selection, or high cost of production. On the basis of our studies concerning the possible use of unmodified PAMAM dendrimers as the scavengers of glucose and carbonyl stress in animal models of human pathology, as well as considering available literature on experimental data of other researchers, we have prepared the brief critical review of the biomedical activities of these unmodified compounds and their most alluring derivatives, especially in the context of possible future perspectives of PAMAMs. Thus, on the pages of this review, we made an attempt to briefly summarize obstacles, emerging from experimental, technical, and human C 2014 Wiley limitations, that may, to some extent, restrain our belief in a brighter future of plain amine-terminated PAMAM dendrimers.  Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:2–14, 2015 Keywords: biomaterials; PAMAM dendrimers; nonenzymatic glycosylation; nanotechnology; nanoparticles; polymers; surface chemistry; toxicity

INTRODUCTION The number of papers concerning the usefulness of PAMAM dendrimers in nanotechnology increases from year to year. Among the various nanoparticles that have become available and are currently being tested, or have been the subject of studies associated with biomedical applications, dendrimers are characterized by unique features because of their small and flexible structures, as well as because of a wide spectrum of their potential use in medicine or technology.1,2 These nanoparticles are usually formulated for targeted delivery to the lymphatic system, brain, arterial walls, lungs, liver, and spleen, and they can be suited to long-term systemic circulation. Considering the above-mentioned concepts, molecular medicine technology, at the nanolevel, may provide novel insights into the diagnosis, treatment, and prevention of various diseases with the use of PAMAMs. In addition, the high number of patents clearly indicates that PAMAM dendrimers are of interest to many scientists and business representatives.3 In 2007, we began to explore the potential antidiabetic applications of PAMAM dendrimers using an animal model of experimental diabetes. In our attempts, both in vitro and in vivo systems were employed to (1) evidence the applicability of PAMAM dendrimers as scavengers of excessive glucose, and to (2) determine their role in preventing the nonenzymatic modifications of biomacromolecules by various metabolic by-products.4–7 Today, based on the bulk of obtained results, we provide an overview of the characteristics of some important factors that are likely Correspondence to: Magdalena Labieniec-Watala (Telephone: +48-42-635-4481; Fax: +48-42-635-44-73; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 104, 2–14 (2015)

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to underlie the burden of easy and direct implementation of PAMAM dendrimers to clinical practice. These include, for instance, the impact of seasonal fluctuations on received experimental data or poor transfer of the outcomes of in vitro studies to in vivo investigations. We also discuss other restrictions that might influence the burden, such as poor understanding of biocompatibility, biodistribution, and biodegradation of PAMAMs, poorly understood pharmacokinetics, their high toxicity, unpredictability in their activity, or high price of commercial dendrimers. These should certainly be taken into account when planning of experiments, collecting data, and drawing conclusions, and more even so, considering the fact that commercial implementation of PAMAM dendrimers is forthcoming. Thus, we indicate that these aspects should be regarded as some restraints in the constructive and reliable conclusions drawn in the course of studying of these compounds. As a peculiar counterweight to plain, unmodified PAMAM dendrimers, we also briefly discuss the potential of their modified derivatives. PAMAM Dendrimers in the Center of Interest of Nanoscientists—Overall Current Status PAMAM dendrimers were, for the first time, synthesized in 1984 by Donald Tomalia, a pioneer chemist in the field of dendrimers.8 Initially, only very few laboratories were interested in studying these polymers. Later, however, researchers have realized that dendrimers have some special properties to offer. During recent years, dendrimers have attracted considerable interest from both scientific and industrial communities. As a result, research efforts of chemists, as well as the other scientists working in the field of dendrimers, increased incredibly, and now a high number of papers and patents concerning these compounds are available.

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As compared with traditional polymers, dendrimers are characterized by significantly improved physical, chemical, and biomedical properties. Such features have made dendrimers very popular all over the world, resulting in their rapid development, thus leading to numerous applications not only in the biomedical area (delivery system, diagnostics, laboratory analytics, therapy, etc.), but also in the industry (cosmetic compositions, synthetic chemistry, dentistic materials, instrumentation, etc.).8 Dendrimers have attracted the attention of researchers interested in drug delivery, in part because of the belief that more precise control over a composition can counter challenges to absorption, distribution, metabolism, excretion, and toxicity. Progress in this field is driven by the belief that polymeric therapeutics will contribute significantly to human health by increasing the efficacy of presently used drugs, as well as by providing opportunities for the use of new agents currently excluded from the clinic because of such restraints, as low solubility and systemic toxicity. Dendrimers provide platforms for drug attachment and have the ability to encapsulate or bind drugs via several mechanisms, such as physical encapsulation, electrostatic interaction, and covalent conjugation. The encapsulation/complexation of drug molecules into/with dendrimers can be widely used in employing different routes of drug administration.9 Currently, it is thought that dendrimers, and among them also PAMAM dendrimers, have a plethora of applications. Hence, it is expected that dendrimers will play a key role in the biomedical sciences of the 21st century. The number of companies involved in the distribution of dendrimers increased dramatically since the early 1990’s of the 20th century. Accordingly, our knowledge of the properties and activities of these compounds has grown incredibly, and each year a number of review articles on this topic is compiled and published.10–12 PAMAM dendrimers, regardless of their toxicity (briefly described below), are still considered “smart agents,” owing to their ability to: (1) participate in the intracellular drug delivery, (2) cross biological membranes/barriers, (3) circulate in a body, and (4) target specific cellular and tissue structures.10 Currently, after nearly 30-year history of the research on PAMAMs, we still search for new possibilities of using them, even despite of the growing number of reports indicating the serious limitations in the utilizing of these agents in a biomedicinal area.13–17 On the basis of the reviewing of the latest papers concerning PAMAM dendrimers, the following challenges have been raised as the most urgent to resolve in order to successfully employ dendrimers in nanotechnology:

r Significant reduction of PAMAMs’ toxicity—strongly der r r r

sirable, i.e. through appropriate terminal groups modifications. Search for the relevance of the outcomes from the in vitro and in vivo studies, regarding dendrimers toxicity. A clear understanding of the interactions between dendrimers and blood components, coagulation process, and fibrinolysis. Recognition of immune responses to dendrimers’ activities and learning of the consequences of such interactions. Resolving of the wide profile of alterations in endothelial cell functioning in order to assess the impact of dendrimers on vessel wall activity.

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r Invention of dendrimer-based drug formulations with relr

atively low clearances, low-plasma half-times, and sufficient degradation profiles in the blood. Elaboration of the most efficient and safest routes of PAMAMs’ administration.

A better understanding of the above aspects of dendrimerbased systems and their pharmaceutics will help to develop a rationale for the design of dendrimers for therapeutic and diagnostic purposes. Nevertheless, in the light of the current knowledge and the progress of research involving animal models, it seems that only some dendrimers, and not necessarily the unmodified PAMAM polymers, have a chance to be used in the treatments of human diseases. It is worth noting that a number of publications and patents places dendrimers lower than quantum dots, fullerenes or carbon nanotubes on a list of nanomaterials of potential interest. On the contrary, it is very difficult to generalize about the near future in an area that is still so progressively developing and includes applications covering such different stages of development.

Limitations and Restraints in PAMAM Applications—Unjustified Fear or Reality? The special architecture and unique behavior of dendritic polymers make them suitable for countless applications in different fields, including adhesives, coatings, catalysts, chemical sensors, or light harvesting materials, to name just a few, but certainly the promise of using dendrimers as drug delivery systems dominated all other fields.18 Although each year flourishes with thousands of papers describing the advantages of the using of PAMAM dendrimers in various areas of science and technology, we have to remember that there are also some disadvantages, which limit the use of these dendrimers and which cast the shadow of doubt on these highly appreciated polymers. First of all, PAMAMs are toxic, and currently this toxicity can only be resolved by modifying the dendrimer structure. Dendritic polymers have great potential in various therapies, especially given their ability to be designed for biological specificity, and therefore their biocompatibility and lack of toxicity are very important qualities. Importantly, however, not all dendrimers are biocompatible or demonstrate low toxicities. As it has been well documented also in our earlier reports, dendrimers carrying cationic groups can exhibit significant toxicity, especially when they are applied at higher doses. Moreover, it should also be stressed here that some investigated dendrimer applications have not used surface-modified counterparts, but plain (non-modified) dendrimers. It has also been the case with our approach. On the contrary, we have to remain aware of the fact that it is just unmodified PAMAM dendrimers that bear the essential and desired chemical characteristics (like the affinity to reactive carbonyl residues). Thus, although using modified PAMAM dendrimers certainly compromises the adverse biological characteristics of these polymers, the presence of unmodified terminal amino groups in the molecule often determines the legitimacy of the use of these compounds as scavengers. Therefore, having reviewed these limitations and restrictions that appeared in our animal model pathology, it should be reconsidered whether cationic PAMAM dendrimers, despite showing some promise in curing hyperglycaemia, may

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have any practical application in the therapy of long-lasting diabetes. Admittedly, rather well-documented scientific knowledge of PAMAMs’ toxicity in an organism is available concerning either long- or short-term PAMAM applications. The oral acute toxicity study described by Thiagarajan et al.19 lasted no longer than 10 days, during which CD-1 mice were given the cationic dendrimer PAMAM G4 at a dose of 50 mg/kg body weight. The maximum tolerated dose of PAMAM dendrimers was established as a function of size and surface functionality, and clinical signs of toxicity were monitored.19 Observations with longer periods of dendrimer administration were conducted by Sadekar and Ghandehari,17 who studied acute, subchronic, and chronic toxicity in male Swiss–Webster mice for 7 days, 30 days, and 6 months. Animals were monitored for routine behavioral abnormalities and changes in body weight.17 Many other papers concerning the in vivo use of PAMAMs in animal models (mice and rats) tend to use higher doses for shorter periods of time, from 2 h to a few weeks.20–24 In general, it looks like the high cost of dendrimers, which strongly influences the overall cost of a study, might have considerably impacted the experimental designs of these studies and supported the rationales of short-term experiments. More importantly however, at least considering this economical aspect, we need to stress that dendrimers are so expensive at present that they discourage longlasting experimental designs, and hence, their potential and widespread use in medical practice nowadays seems more an illusion than reality. We should emphasize here that, at least with regard to our approach of using PAMAM dendrimers as the hypoglycaemizing agents, one might rather unlikely reasonably consider using such costly compounds for a long treatment period, keeping in mind that the treatment of people suffering from diabetes can last many years or even decades. The next area receiving a lot of attention is dendrimer biodistribution, bioavailability, biodegradation, and pharmacokinetics. Fundamental pharmacokinetics and biodistribution are principally based on anatomical and physiological characteristics, as well as, not to a lesser extent, on the physicochemical properties and mutual interactions of a variety of (macro)molecules of biological origin. It is because of such interactions that dendrimers administered to an organism rather quickly penetrate into the bloodstream, and then become gradually eliminated from the blood pool and distributed to particular organs for disposition, where their efflux and elimination from tissue remain very low.25 Some in vivo experimental attempts have been performed to learn the persistence of dendrimers in the organism and to determine where dendrimers are accumulated in the course of experiments. The findings, originating from in vivo studies of laboratory animals, on biodistribution, biodegradation, and elimination of plain (unmodified) and surface-modified PAMAM dendrimers are briefly summarized in Table 1. These data strongly suggest that the biodistribution properties of dendrimers should certainly be more extensively studied and explained in much more considerable detail before dendrimers have a chance to be used in treating human diseases.24,26–28 Although the number of publications on dendrimer research, especially those concerning potential applications of dendrimers in medicine, has gradually increased over the last two decades, only a few dendrimers are currently tested in the ongoing clinical trials. It should always be remembered that medical applications will require official approval by the rele-

vant administrative institutions (e.g., the US FDA—Food and Drug Administration in the US) before being commercially implemented. In July, 2003, the FDA allowed for the first clinTM ical trials of a dendrimer-based pharmaceutical—Vivagel ,a topical microbicide for the prevention of HIV infection in women, developed by the Australian company Starpharma. Another dendrimer undergoing preclinical study is the multiantigenic peptide PHSCN-lysine dendrimer, applied in a metastatic murine cancer model in an attempt to inhibit the invasion and growth of breast cancer cells via the mechanism of the "5 $1 integrin-selective recognition.42 In 2006, the FDA launched a Nanotechnology Task Force for critical regulatory issues regarding nanomaterials containing drug products. Accordingly, guidelines for IND (Investigational New Drug Application) are being developed to address specific issues of nanomaterials containing drug products, which have physicochemical properties unique or distinct from drug molecules. This highlights the importance of biodistribution and pharmacokinetics as a topic of the assessment of absorption, distribution, metabolism, and excretion. Moreover, the FDA recommends a long-term study as a standard guideline to assess their safety and efficacy. As reported in the literature, dendrimers such as PAMAM are well tolerated in mice without any serious toxicity over time.43,44 However, some vacuolization of the cytoplasm in the liver has been observed after long-term administration of cationic PAMAM dendrimers G3–7 at 2.5–10 mg/kg.24 It is important to consider not only the properties of the dendrimer or dendrimerbased drug, but also the experimental design for careful evaluation and interpretation of pharmacokinetic and biodistribution studies with regard to safety and efficacy during a possible clinical use. There is still another issue, which certainly should not be underestimated. A considerable inconsistency and/or variability of outcomes originating from studies on animals may be simply because of the diversity of the used animal models and strains, differentiated treatment durations, and/or the phenomenon of seasonal fluctuations. It has been noticed and emphasized earlier45–48 that when working with laboratory animals, the phenomenon of seasonal fluctuations should certainly be taken into consideration, as such fluctuations can potentially affect animal metabolism and bioenergetics, also under a standard housing regime.49,50 Another important point concerns the use of different laboratory rodent strains to study similar models of pathology. Some strains may demonstrate differentiated sensitivity to the tested compounds or may undergo faster adaptation to their possibly harmful impacts.45–47,51 Consequently, the outcomes gathered from studies conducted on various strains of animals, or even on animals provided by different distributors,52 may differ considerably, which in turn, may influence data interpretation and further formulation of conclusions. Overall, testing of the pharmacological or toxicity profiles in laboratory animals may be associated with some confounders, such as seasonality or animal strain and origin, that are likely to considerably affect the perception of “true” facts. Modified Versus Unmodified Dendrimers—What Is the Future for Plain Unmodified Dendrimers in Biomedical Applications? One specific way to overcome the obstacles associated with the toxicity and/or other undesired side effects encountered when using polycationic PAMAM dendrimers and to achieve the expected improved therapeutic effects through enhanced

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Cationic PAMAM dendrimers (G3 and G4); anionic PAMAM dendrimers (G2.5, G3.5, and G5.5)

Cationic PAMAM dendrimers G3 and G4

Cationic PAMAM dendrimers, G4 and G5

Cationic PAMAM dendrimer G4 Anionic PAMAM dendrimer G6.5

Cationic PAMAM G4 dendrimers (Gd-radioactive labeled) Cationic PAMAM dendrimers: G3–G6 (Gd-radioactive labeled)

Unmodified (plain) Cationic PAMAM dendrimers: G3, G5, G7

Wistar rats

Female BALB/c healthy and BALB/c athymic nude mice Wistar rats

Female CD-1 mice

Mice

Anionic dendrimers show rapid serosal transfer rates and have low-tissue deposition. Generation 5.5 displayed higher tissue accumulation than G2.5 and G3.5. In contrast, cationic PAMAMs showed much higher tissue accumulation, with lower transport rates

Significant accumulation in liver

Dendrimers were found in blood, kidneys, liver, and spleen

Penetration of PAMAM G4 into brain parenchyma and different neural layers was evidenced Dendrimers were present in liver, kidneys, lungs, blood, and urine

Dendrimers were present in kidneys, liver, intestine, muscles, blood, and urinary bladder; their distribution and levels in these organs were not tissue-specifically dependent

Female nude mice

Nude mice

G3 showed the highest accumulation in liver, kidneys and spleen. G5 was detected at the highest concentrations in spleen, intestinal tissues, and pancreas. The highest levels of G7 were present in urine and pancreas, also detected in blood, lungs, kidneys, liver, and heart tissue Liver, kidneys and blood

Biodistribution

Male Swiss–Webster mice

Biological Material

Biological Fate—Characterization

Dendrimers were readily cleared from the circulation. Only 0.1%–1% of the recovered dose was detected in blood at 1 h No data available

The major amounts of the bioavailable dose of the dendrimer were excreted in the urine by 4 h No data available

10%–13% of the injected G6, G5, and G4 dendrimers and 25% of G3 dendrimers were excreted within 2 days; up to 70%–82% of administered dendrimers were excreted in urine No data available

Urinary excretion at 48 h after injection

No data available

Biodegradation/Elimination

Biodistribution and Biodegradation/Elimination of Unmodified and Modified PAMAM Dendrimers Recorded in the In Vivo Studies

Type of PAMAM Dendrimers

Table 1.

Continued

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Wistar rats

Rabbit model of cerebral palsy

Cationic PAMAM dendrimers G3 and G4

Neutral PAMAM G dendrimer G4

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Hydroxyl-terminated PAMAM dendrimer G4

Iodobenzoate– biotinylated–PAMAM dendrimers G0–G4 Acetylated PAMAM dendrimers G5 modified

Female New Zealand white rabbits

Female nude mice

Athymic mice

Female nude mice

Wistar rats

Cationic PAMAM dendrimers: G0–G3

Modified PAMAM dendrimers G5–G7, COOH terminated (anionic dendrimers)

Wistar rats

Biological Material

Cationic PAMAM dendrimers: G0–G3

Type of PAMAM Dendrimers

Table 1.

Dendrimer accumulation was shown in kidneys, liver, lungs, heart, brain, blood, and urea at 24 h after the injection. The highest levels of the dendrimer were present in kidneys, lungs, and liver. Very low doses of the dendrimer were present in the brain

G5 - predominant accumulation (80%) in kidneys after 30 min of administration; G6 - high levels in kidneys and liver (up to 25%) during the first 30 min; G7 - the highest plasma circulation time, but this dendrimer was also uptaken by liver and kidneys in a nonspecific way High accumulation in kidneys and liver; the accumulation in kidneys increased significantly with the increasing generation and positive charge Dendrimers were found in blood, kidneys, liver, spleen, brain, pancreas, and heart

All the tested dendrimers were absorbed by nasal tissues, with low membrane damages caused by higher dendrimer generations; the third-generation of dendrimers effectively improved the nasal absorption of drugs, such as insulin, calcitonin, and dextran All the tested dendrimers were absorbed by lung tissues without any signs of toxicity. PAMAM G3 was the most effective in the increasing of the pulmonary absorption of insulin and calcitonin, showing no membrane damage Both the tested dendrimers revealed the ability to penetrate to deep layers of the skin. It was also proven that these dendrimers can improve the transdermal delivery of other agents (i.e., drugs) in a concentration-dependent manner (G4>G3). Dendrimers were found in activated microglia and astrocytes

Biodistribution

Biological Fate—Characterization

Circulation time in the blood depended on dendrimer generation; anionic dendrimers circulated longer than their cationic counterparts Quickly cleared from the blood; blood levels of 0.13%–0.2% dose/g 4 h after administration Similar patterns of clearance were observed for the heart, pancreas, and spleen. The level of this dendrimer in the kidneys decreased rapidly and was maintained at a moderate level over the next several days; the concentration of the dendrimer in the brain was low More than 90% of the injected dendrimer dose was cleared out from the rabbits over 24 h, with less than 5% in a blood circulation

No data available

No data available

No data available

No data available

Biodegradation/Elimination

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biocompatibility, is to chemically modify their surface. Although a dendrimer’s cytotoxicity is often dependent on the core chemistry, their interactions with cellular and subcellular structures are most strongly influenced by the nature of the dendrimer surface. The critical review of a literature provides a rational survey on what has been carried out regarding this problem. The cytotoxicity of cationic PAMAM dendrimers has been attributed to their interactions with negatively charged cell surfaces.23,24 As the biological safety of using unmodified PAMAM dendrimers has been questioned, efforts are now being made to modify dendrimer surface groups by conjugating them with either fatty acids, carbohydrates, or polyethyleneglycol (PEG).53 The conjugation with PEG chains has been considered as the method of choice for reducing PAMAM toxicity and for increasing the biocompatibility of the dendrimers, mainly because PEG is nontoxic, nonimmunogenic, and has a favorable pharmacokinetic profile, as well as advantageous tissue distribution.54 In particular, the in vitro experiments performed with using various human and animal cell lines have shown that increasing the coverage with PEG chains of amino-terminated PAMAM results in the decreased cytotoxicity of these dendrimers.55,56 In addition, PEGylation of PAMAM dendrimers has also been reported to greatly prolong the circulation half-life and improve biodistribution and biocompatibility.55,57 Moreover, Wang et al.58 observed that whereas plain PAMAMs were able to stimulate the overproduction of reactive oxygen species and to disturb the function of mitochondria, consequently leading to cell death, their PEG-modified counterparts inhibited these phenomena. When the strategy of dendrimer surface modifications became more familiar, other modifying chemicals were used for this purpose. It was noted that covalent attachment of acetyl groups59 and lauroyl groups53 to the peripheral amino groups of PAMAM dendrimers decreased their cytotoxicity toward host cells, probably because of the reduction of the number of protonated amino groups and shielding of the positive charges on the dendrimers’ surface. It is also worth noting that PAMAM acetylation has been found to increase their ability of delivering siRNA to tumor cells.60 Other efforts to modify PAMAM dendrimers for gene or siRNA delivery have also been successful in decreasing dendrimer cytotoxicity and increasing their efficiency in DNA delivery.61 Such modified dendrimers have become effectively used in several nanomedical applications. Not only they bear the advantage of improved stereogeometry and spatial structure, but are also characterized by considerably reduced toxic activity. It has been proven that the biological profile of a modified dendrimer is likely to be different from that of an unmodified dendrimer. Ciolkowski et al.62 observed that a fourth generation of PAMAM dendrimer modified by pyrrolidone residues demonstrates no considerable hemolytic activity and shows merely minor cytotoxic effects, as compared with its “parent” counterpart. In addition, its reactivity and ability to bind to human serum albumin also became reduced, compared to the unmodified PAMAM G4 molecules.62 Interestingly, in all the above-mentioned approaches, rather identical conceptual strategy was implied, followed by identical consequences: the apparent loss of surface-reactive amino groups. Thus, the same very challenging feature in the chemical structure of polyamine-terminated PAMAM dendrimers that once made our attention focused on a variety of putative applications of these multivalent nanoparticles, that is, their electrophilic surface, have vanished at the cost of diminished DOI 10.1002/jps.24222

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cytotoxicity. Considering both ends of this story, it may seem at the first sight that struggling for a considerable reduction of undesired polycationicity and maintaining at the same time desirable reactivity of PAMAMs may lead to no reasonable solution of this drawback. As far as amine group modification may decrease the overall polycationicity, and thus may even reduce the burden of PAMAM’s toxicity, the same modification may inevitably lead to diminished reactivity and hence, largely suppress attractiveness of these poly-amine-terminated compounds. Apart from their biomedical activity (studied especially in the in vitro models), there is another interesting aspect regarding the modified dendrimers, which concerns their cellular and tissue uptakes (of interest also in the in vivo investigations). The studies undertaken to elucidate the mechanisms and pathways of dendrimer internalization and trafficking are important, as they may help in defining how surface modifications of plain polymers may enhance the cellular uptake of nanoparticulate dendrimer–drug complexes and how they may potentially increase drug delivery across physiological cell barriers. The study of Saovapakhiran et al.63 provided the evidence that the surface properties of dendrimers influenced the initial mode of dendrimer internalization in HT-29 cells. The authors claim that the modification of the parent G3 dendrimer with lauroyl moieties increases the rate of intracellular uptake, which might account for the observed increase in the rate of oral drug delivery in biomedical systems. In addition, the modification of nanoparticles with lauroyl moieties appeared to reduce the extent of lysosomal accumulation, thus suggesting potentially lesser exposure of the dendrimer conjugate to the highly acidic lysosomal environment and hydrolytic enzymes, a potentially important issue when considering delivery of acid-labile drugs or drug conjugates. Thus, dendrimer modification with lauroyl chains has become attractive, as it has been proven that such a modification enhances the internalization kinetics of the modified dendrimer into human intestinal epithelial cells. This may potentially improve drug delivery via the oral route of administration.63 Nevertheless, apart from these promising results, there is a relative scarcity of literature reports describing and referring to the differences/dissimilarities in cell membrane transport, biodistribution, and activity between modified and unmodified PAMAMs. Coming back to the question raised in the title of this subchapter, we are faced with a choice of plain or unmodified PAMAM dendrimers. On one hand, based on our data accumulated over the last 4–5 years, one might anticipate that unmodified PAMAM dendrimers should probably not be seriously regarded at present as safe and useful pharmaceuticals, and thus should not be unrestrainedly considered as “drugs” directly used in the chronic treatment of metabolic disorders induced by excessive glucose. Their unpredictable biological activity, as well as the high potency of PAMAM dendrimers to trigger nonspecific, difficult to control, and often toxic side effects should encourage researchers to verify and validate various possible pathways and mechanisms of their toxicity, especially when such effects may be disguised by the “mask” of therapeutic effect. On the contrary, despite the scarcity of available data on modified PAMAM dendrimers, it may be argued that this class of dendrimers looks very attractive, promising, and challenging in the area of nanomedicine. The initial positive feedback from new endeavors in dendrimer chemistry and the observed improved biomedical characteristics of novel and “useful”

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dendrimer modifications has triggered a veritable avalanche of new ideas in recent years. These modifications, which have given rise to a new generation of safe and effective dendrimers, have been demonstrated by numerous investigators to exhibit much reduced overall toxicity and markedly enhanced efficiency, improved biodistribution, and bioavailability. Although further studies are necessary to examine their detailed mechanisms of action, intracellular/body distribution, and clinical safety as propharmaceuticals with a potential utilization in biomedical area, we may conclude at present that modified PAMAM dendrimers with much improved pharmacological profiles promise a much brighter future in comparison with their antecedent molecules. GlycoPAMAM Dendrimers and Glycosylated PAMAM Dendrimers —Are They the Same Agents in a Different Guise? In recent years, it has become overwhelmingly clear that sugars play a key role in biological processes. Because of their high biocompatibility and biodegradability, carbohydrate-based materials have also been widely used for pharmaceutical and medical applications. It soon became apparent that synthetic carbohydrate polymers—the so-called “glycopolymers”—can also exhibit specific interactions with proteins, and hence, one can assume that a plethora of biological interactions of natural carbohydrates can be mimicked by synthetic polymers containing sugar moieties64 (Fig. 1). The first glycodendrimers described in the literature were reported in 1993 by Roy et al.,65 and since then the field of glycodendrimers has matured and expanded to quite an unprecedented level. It is worth noting that glycodendrimers were initially designed as bioisosteres of cellsurface multiantennary glycans, but it was soon after that the scientific community became inspired by the potential biological and biomedical applications of this class of dendrimers. This, in turn, triggered further investigations and the gradual development of new synthetic strategies leading to these nanostructures.66 Since then, we have witnessed considerable progress, mainly in such fields of highly advanced chemistry, as organometallic chemistry, chemoenzymatic catalysis, silicon chemistry, and dendrimer self-assembly. An excellent review on the synthesis of glycodendrimers was published in 2010 by Chabre and Roy,67 ; therefore, we do not intend to repeat these details on the synthesis in this paragraph.

Given their commercial accessibility, PAMAMs have been the scaffolds most widely used in modern investigations. PAMAMbased dendrimers, having built-in amine functionalities on their surfaces, became the first and most frequently used scaffolds for sugar attachment. They have been modified with a large variety of sugar derivatives and with varied sugar densities. Therefore, in the toxicity evaluations, they certainly represent the most deeply studied scaffolds. Importantly, scaffolded glycodendrimers were shown to be nonimmunogenic, a key property if they are to be used as bacterial or viral antiadhesins.68 The very first example of saccharide-substituted PAMAM dendrimers was proposed by Okada and Aoi,69 who described the synthesis of the so-called “sugar balls” via an amide-bond formation starting from sugar lactones. Although this process constituted a straightforward manipulation, it suffered from the disadvantage of sacrificing the reducing sugars, which also served as extended linkers.70 Then, several other strategies have also been used in order to functionalize PAMAM dendrimers with carbohydrates, involving: (1) introduction of thiourea linkages formed by treating of amino dendrimers with isothiocyanated saccharide derivatives, (2) direct amide linkages with sugars-bearing carboxylated or activated ester derivatives, (3) reductive amination, or (4) incorporation of chloro- or bromoacetamide groups onto PAMAM dendrimers or saccharides to afford highly electrophilic species that can react, for instance, with thioderivatives or aminoderivatives.66 Moreover, saccharide moieties can be localized in the center of the dendrimer, as a part of its core71,72 ; they can build dendrimer side branches73 or may cover the outer surface of dendrimers after attachment to terminal groups.53,74 A plethora of carbohydrates, including chitosan,75 carboxymethylchitosan,76 heparin,77 galactose, glucose, mannose,78 or lactose,79 allows to transform the structure of a given molecule in order to be adapted to the needs of a researcher, and to give a great potential for its use in a variety of applications. Today, glycodendrimers represent a group of biopolymers with fascinating properties. On the basis of the reports concerning glycodendrimers, it appears clear that such prepared compounds possess several desirable properties, making them good candidates for a plethora of biological applications. Most dendrimers show good to excellent hydrophilicity that allows them to adhere nonspecifically to the typical microtiter plates used in conventional ELISA assays, which further makes them possible

Figure 1. Schematic chemical structures of glycodendrimers, glycosylated dendrimers, and N-glycosylated (glycated) PAMAM dendrimers. Surface functional groups may be modified with various residues (balls), involving mono- or oligosaccharides. Labieniec-Watala and Watala, JOURNAL OF PHARMACEUTICAL SCIENCES 104:2–14, 2015

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coating antigens. Some glycodendrimers have been constructed on scaffolds that enable them to incorporate complexes useful for drug targeting. Several glycodendrimers, particularly those having mannoside or oligomannoside end groups, have the potential to become entirely synthetic vaccines. Noticeable are the examples directed at raising the protective immune responses against HIV-1 infection. Glycodendrimers bearing some cancer markers, such as the sialyl Tn antigen [STn antigen; Nacetylgalactosamine (GalNAc), linked to serine or threonine by a glycoside bond] and the Thomsen–Friedenreich antigen [TF antigen or T antigen, formed by the substitution with galactose (Gal($1–3)GalNAc)], can elicit potent immune responses, if properly conjugated to immunogenic T cell peptides recognized by MHC molecules. There are numerous examples of glycodendrimers being autoassembled, using a variety of transition metal chelating groups.80 The most appealing applications seemed to belong to bacterial and viral antiadhesins. Several successful case studies have unambiguously demonstrated their potential to block the corresponding receptors of these proteins. The most obvious applications are those involving urinary tract, gastrointestinal, or pulmonary infections. Certain glycodendrimers have shown IC50 values in the nanomolar range, which makes them good drug candidates. Overall, the future looks extremely promising for glycodendrimers and yet there is still a plenty of unexplored activities.68,81–84 Apart from “glycodendrimers”, another class of dendrimers, named “glycosylated dendrimers” has been distinguished. In contrast to glycodendrimers, glycosylated dendrimers are usually formed from anionic PAMAM dendrimers (half-generation PAMAMs), whose surfaces are modified with glucosamine moieties85 (Fig. 1). Nevertheless, both terms concern similar groups of compounds, which are characterized by similar properties, and both types are synthesized to obtain dendrimers, which may be better suited to various biomedical applications than their unmodified counterparts. In the case of our studies, we use the term “N-glycosylated dendrimers” or “glycated dendrimers” for dendrimers formed as a final product in the process of nonenzymatic glycosylation (N-glycosylation, glycation), which has been characterized as a driving force for protein glycation. In our previous report, we demonstrated in the in vitro study that plain fullgeneration PAMAM dendrimers are able to react with glucose molecules and to form stable bonds with them.7 On the basis of these findings, we have evidenced that the nonenzymatic reaction between the glucose and amine terminal groups of dendrimers may underlie the formation of glycated/N-glycosylated dendrimers. In this way, PAMAM dendrimers can act as glucose scavengers during hyperglycaemia, finally resulting in reduced protein glycation and glycooxidation. Hence, contrary to other papers concerning the area of the interactions between dendrimers and saccharide molecules, we propose quite a different mechanism for obtaining glycosylated dendrimers: not one that acts in the course of a well-controlled chemical synthesis, but rather via spontaneous reactions, which can occur on the dendrimer surface under conditions of overload with free glucose. In this context, thus obtained N-glycosylated dendrimers have to be regarded only as final by-products of nonenzymatic N-glycosylation, and not as a result of a designed and oriented chemical synthesis, the outcomes of which could become a source of potential applications in many branches of biomedicine. Noteworthy, despite the detailed chemical mechanisms of their formation, both glycodendrimers and various glyDOI 10.1002/jps.24222

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cosylated dendrimers, regardless of whether generated in the course of designed chemical synthesis or spontaneously, in the presence of excessive glucose, have one common and unifying characteristic. They are shielded by sugar moieties, which make them more hydrophilic and less cytotoxic. Hence, they are also better oriented to transmembrane transport or intercellular interactions. At this point, another interesting anticipation could be made. Considering this, we may easily imagine that primarily toxic plain PAMAM dendrimers may gradually become safer and “more friendly” toward cellular environment upon undergoing nonenzymatic N-glycosylation in the presence of excessive glucose (like in diabetes or under conditions of severe hyperglycaemia). Indeed, we have demonstrated such a phenomenon in our experimental model of streptozotocin-induced diabetes in rats: the higher extents of hyperglycaemia in diabetic animals appeared more protective toward the observed PAMAM toxicity.5 PAMAM Dendrimers Modified with Lysine—The Dawning Area for Therapeutic Applications or Another Illusion? The outcomes of our in vivo studies carried out with unmodified PAMAM dendrimers lead to the conclusion that PAMAMs have, in general, the chemical properties suitable for their consideration as hypoglycaemizing and antiglycation agents. Although the hallmarks of severe diabetic hyperglycaemia have not been completely reduced compared with the values observed in nondiabetic counterparts, these polyamine-terminated polymers appeared to bear the potential to limit some consequences of long-lasting diabetes originating from chronic states of excessive glucose. Importantly however, some burden of adverse toxic effects is also associated with the use of plain PAMAM dendrimers. Rather unpredictable biological activity of plain fullgeneration PAMAM dendrimers, as well as a variety of toxic side effects that may be triggered by these nanocompounds, certainly presents new challenges for researchers toward better understanding of pharmacokinetic profiles and possible mechanisms of interaction of these polycationic polymers with target tissues. Nevertheless, it is certainly not our intention to present this group of compounds in an exclusively bad light and to pigeonhole the initial idea of using them as scavengers of excessive glucose as a dead end or ineffective target. In our opinion, the slight modifications of amino termini on PAMAM’s surface could be a good option for applying them in curing metabolic impairments of diabetes. The new, purely theoretical concept as yet assumes that it seems plausible to bind lysine residues to the plain amino-terminated PAMAMs in order to receive “polylysylated” PAMAM dendrimers (Fig. 2a). As such dendrimers acquire two new free amino “valencies” of lysine (" and g) per one occupied primary amino group on the dendrimer’s surface, it may reasonably be anticipated that PAMAM dendrimers modified with L-lysine should be even more effective in the scavenging of excessive glucose than their unmodified counterparts. As the process of nonenzymatic glycosylation takes place exactly at the g amino groups of protein lysine residues upon the interactions with glucose,86 it can be presumed that the PAMAM dendrimer molecules loaded with lysine residues on their surfaces could be an ideal and effective target in reducing of the postenzymatic modifications of proteins, and theoretically even more suitable for such a scavenging than nonlysylated dendrimers. In particular, the amino groups of terminal

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Figure 2. Schematic chemical structures of poly(L-lysylated) PAMAM dendrimers (a) and PLL dendrimers (b). In the hypothetical structure of poly(L-lysylated) PAMAM dendrimers, the residues of L-lysine (balls) may be linked to surface terminal groups either directly (via peptide bond) or with the use of a linker.

lysyl residues in cationic “polylysylated” PAMAM dendrimers are considered as very promising moieties for the attachment of other molecules and different functional groups. As the density of the terminal amino groups on the dendrimer’s surface increases with increasing generations of PAMAM, it has been suggested that the attachment of other molecules to the polymer becomes easier.87 In fact, the idea of using lysine derivatives for the modulation of various cellular and subcellular events has quite a long history in scientific research. Thanks to the above-mentioned advantages, lysine-containing dendrimers have already attracted the attention of researchers over three decades ago and they have gained a “second look” over time as potentially more effective drug carriers. The growing interest in poly-L-lysine (PLL) dendrimers has triggered a new wave of investigations in the hope for their possible more successful application in biomedical nanotechnology. Indeed, in recent years, the increasing interest in the development of various types of dendrimers has led to the invention of several lysine-based dendrimers. Earlier, a linear polylysine was employed for many years as a vector for the cellular delivery of DNA.88 Currently, PLL dendrimers, first prepared in the early 1980s, are a well-established family of dendrimers89 (Fig. 2b). Though less commonly reported than PAMAM dendrimers or the poly(propylene imine) dendrimers, they revealed their potential usefulness in various biological applications, such as gene delivery agents, drug carriers, peptide antigens, vaccines, and antimicrobial agents.90 However, PLL dendrimers are commonly considered both cytotoxic and haemolytic, in that they bear numerous surface lysines, and thus they still display strong polycationic characteristics. Therefore, the investigators of PPL most often make attempts to reduce these potential adverse effects and to modify the surface of these dendrimers with nonionic groups, such as PEG or other chemical residues (phenyldicarboxylate, naphthylsulfonate, arylsulfonate, succinate, fullerene, or arginine

groups). The problem has been rather extensively studied and we are aware now that the capping the surface of PLL dendrimers with these groups reduces vascular binding and increases metabolic stability, compared with the corresponding cationic PLL polymers.27,91,92 It has also been shown that such modifications improve the efficacy of a chemotherapeutic drugs in the cancer treatment, when PLL dendrimers are employed as drug carriers.93 Moreover, these polymer modifications have proven to effectively prolong blood circulation time, increase tumor accumulation, and protect their drug cargos from enzymatic access.94 Thus, it seems that dendrimers based on PLL residues are promising candidates, next to PAMAMs, for their use in different biomedical applications. However, there is still the need to keep in mind the fact that their utility may be limited by proteolytic instability, interactions with endothelial surfaces or blood components, or a capture by the phagocytic cells of the reticuloendothelial system. Importantly, it was also observed in the in vivo study that uncapped PPL dendrimers undergo extensive degradation to monomeric lysine.95 Although many of these limitations may be overcome by conjugations with the chemical groups mentioned above, the use of plain PLL dendrimers, analogically as in the case of plain PAMAMs, raises doubts. Despite some restraints, potentially new properties of PLL dendrimers have been readily verified experimentally. For instance, Sideratou et al.96 have revealed in their in vitro studies that arginine end-functionalized PLL dendrimers efficiently protect insulin against enzymatic degradation by trypsin and "-chymotrypsin. Furthermore, insulin release rates have been found to be strongly related to the number of arginine end groups. The authors postulated that insulin oral administration to systemic circulation may thus be more attractive than injection, although oral supplementation is still hindered by several obstacles, such as proteolytic enzymes, sharp pH gradients, and low epithelial permeability. Hence, it was

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decided to make a complex of insulin with biodegradable arginine end-functionalized PLL dendrigrafts and investigate the stabilization of complexed insulin against enzymatic degradation by trypsin and "-chymotrypsin, as well as its release in enzyme-free simulated intestinal fluid. Finally, the authors have demonstrated and proven that modified PLL dendrimers offer their new biomedical application as safe insulin delivery systems.96 Overall, our critical look into the accumulated literature evidence concerning PLL dendrimers inevitably leads to the conclusion that without further extensive studies we are still at the crossroads of uncertainty as to whether unmodified/modified PLL or PAMAMs modified by lysine residues have a chance of the successful replacing of plain PAMAMs, when we consider using them in future in the therapy of diabetic hyperglycaemia. On one hand, we may be embraced with their encouraging antiglycation and anticarbonylation potential, on the other hand, we have to realize that most likely we would deal with the adverse side effects of these agents. As in the case of plain amine-terminated PAMAMs, we may be “forced” to modify their structures appropriately. However, we are aware that such a modifying of polycationic PLL or lysylated PAMAM dendrimers’ surface in order to minimize any adverse side effects may be a two-edged sword, very much alike to that which we considered with respect plain PAMAMs. Although we are now well prepared to perform such modifications to compromise dendrimers’ toxicity, it is always at the cost of less efficient scavenging of excessive glucose. In general, even though the idea of the application of dendrimers as hypoglycaemizing agents have once seemed an intriguing challenge at the first sight, the experimental evidence accumulated hitherto has revealed some potential hindrances and obstacles that might be encountered with this approach in a practice. On the contrary, rather a big load of incentives for further research on how to overcome such obstacles (i.e., by modifying a way of their administration) lies in a supposedly great potential of lysine-containing dendrimers in “scavenging” the excessive electrophiles, as is the case under conditions of glucose and/or carbonyl stress.

r

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on key organs for organism functionality (liver, kidneys, heart, and brain) should be carefully examined. Although the number of laboratories conducting in vivo studies with dendrimers is gradually increasing, the most appropriate (nontoxic and effective) way of dendrimer administration still needs to be developed. As a result of the recent efforts for evaluating the most successful method of dendrimer delivery to organism, we consider the development and verification new modes of dendrimer administration to be of the utmost importance, highlighting that the route of dendrimer delivery to target sites has a great influence on the quality of therapy. A recently emerging problem with the interpretation of the results gained in various seasons and originating from different animal strains/breeds and models should note the necessary attention paid to careful design and further conducting of experiments with animals. This phenomenon needs to be further explored. Most of the conclusions regarding the properties and activity of dendrimers is derived from the outcomes of in vitro studies. Unfortunately, the outcomes collected from the in vitro and in vivo studies correlate poorly; hence, further investigations should be carried out in order to explain and further exploit this tendency. Although numerous scientific reports seem encouraging and raise optimism for the potential use of dendrimers in a variety of medical applications, their high cost of the production and, consequently, their high price, may seriously limit the use of dendrimers in the treatment of long-lasting diseases (i.e., diabetes).

ACKNOWLEDGMENTS This work was supported by the grants from the Ministry of Science and Higher Education, N N401 001236 and N N405 261037. The authors declare no conflict of interests.

REFERENCES CONCLUSIONS To fully exploit the putatively tremendous potential of PAMAM dendrimers in biomedicine, a detailed understanding of a variety of their properties is needed. In our opinion, the list of the following aspects can be spotlighted as focus points for further considerations:

r Despite the extensive efforts and the progress achieved so

r

far, the problem of a reliable method of evaluating nontoxic doses of the tested generations of PAMAM dendrimers still remains underdeveloped. Thus, the main objective for future research should be the estimation of optimal doses, at which dendrimers could be applied with the required effectiveness. The toxicity of cationic dendrimers is still the major problem obstructing their preclinical and clinical applications. It has been repeatedly reported that dendrimers can induce irreversible damage to organs, cells, and tissues in tested organisms (mainly tested in rats and mice). Thus, the impact of dendrimers on overall animal survival and

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Labieniec-Watala and Watala, JOURNAL OF PHARMACEUTICAL SCIENCES 104:2–14, 2015

DOI 10.1002/jps.24222