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International Journal of Pharmaceutics 464 (2014) 152–167

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

j ourna l h om epa ge: www.elsev ier .com/ locate / i jpharm

harmaceutical Nanotechnology

an metabolic impairments in experimental diabetes be cured witholy(amido)amine (PAMAM) G4 dendrimers? – In the search forinimizing of the adverse effects of PAMAM administration

agdalena Labieniec-Watalaa,∗, Tomasz Przygodzkib, Katarina Sebekovac,ezary Watalab

University of Lodz, Faculty of Biology and Environmental Protection, Department of Thermobiology, Pomorska 141/143, 90-236 Lodz, PolandDepartment of Haemostasis and Haemostatic Disorders, Medical University of Lodz, University Clinical Hospital No. 2, Zeromskiego 113, 90-549 Lodz,olandInstitute of Molecular BioMedicine, Comenius University, 811 08 Bratislava, Slovakia

r t i c l e i n f o

rticle history:eceived 14 October 2013eceived in revised form 4 January 2014ccepted 7 January 2014vailable online 21 January 2014

eywords:AMAM dendrimerstreptozotocin-diabetesurvival

a b s t r a c t

Poly(amido)amine (PAMAM) G4 dendrimers, given intraperitoneally to diabetic rats, have been reportedto scavenge excessive blood glucose and minimize the effects of hyperglycaemia, however, at the cost ofreduced survival. This paper is the first to compare the effectiveness of three different routes of PAMAMG4 administration with regard to minimizing the adverse effects of hyperglycaemia in rats. Hence, the aimof the study is to identify the most effective and the least harmful method of dendrimer administration.Control and streptozotocin-diabetic Sprague-Dawley rats were exposed to PAMAM G4 (0.5 �mol/kg b.w.)for 60 days, administered intraperitoneally, intragastrically or subcutaneously.

Intraperitoneal and subcutaneous administration of PAMAM G4 was found to be most effective insuppressing the long-term markers of hyperglycaemia, while the intragastric route appeared the least

iabetic hyperglycaemia markersprague-Dawley ratsn vivo study

effective. Otherwise, the greatest incidence of adverse effects was associated with intraperitoneal andthe lowest with subcutaneous delivery. Harmful effects of intragastrical administration were much lowercompared to intraperitoneal route, but at the cost of reduced hypoglycaemizing potential. Otherwise,subcutaneous injection represents the best compromise of moderate PAMAM dendrimer toxicity andeffective reduction in the markers of long-term severe hyperglycaemia in chronic experimental diabetes.

Abbreviations: CMG, control animals given methanol (vehicle) intragastri-ally; CMI, control animals given methanol (vehicle) intraperitoneally; CPG, controlnimals given PAMAM intragastrically; CPI, control animals given PAMAM intraperi-oneally; DMG, diabetic animals given methanol intragastrically; DMI, diabeticnimals given methanol intraperitoneally; DMS, diabetic animals given methanolubcutaneously; DPG, diabetic animals given PAMAM intragastrically; DPI, diabeticnimals given PAMAM intraperitoneally; DPS, diabetic animals given PAMAM sub-utaneously; GRAN, granulocytes; Hb, haemoglobin; LYMPH, lymphocytes; MCV,ean corpuscular volume; MCH, mean cell haemoglobin; MCHC, mean corpuscu-

ar haemoglobin concentration; MID, minimum inhibitory dilution a measure ofare cells and a number of precursor white cells; MPV, mean platelet volume; PCT,lateletcrit; PLT, platelet count; STZ, streptozotocin; WBC, white blood cells.∗ Corresponding author at: University of Lodz, Department of Thermobiology,omorska 141/143, 90-236 Lodz, Poland. Tel.: +48 42 635 44 81;ax: +48 42 635 44 73.

E-mail addresses: magdalab@biol.uni.lodz.pl (M. Labieniec-Watala),omasz.przygodzki@umed.lodz.pl (T. Przygodzki), katarina.sebekova@szu.skK. Sebekova), cezary.watala@umed.lodz.pl (C. Watala).

378-5173/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijpharm.2014.01.011

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

The potential application of poly(amido)amine (PAMAM) den-drimers seems very promising, however, both the in vitro and in vivotoxicity profiles of these agents is nowadays considered a majorconcern. In general, dendrimers are hemolytic and cytotoxic, andthese disadvantageous characteristics depend on both the molec-ular weight (generation) and on the number and type of terminalgroups on their surface (Domanski et al., 2004; Mukherjee et al.,2010; Prieto et al., 2011).

Reports dealing with toxicity of PAMAM dendrimers are largelyinconsistent or even contradictory in claiming either considerable,slight or even no adverse side-effects. The majority of studies onPAMAM dendrimers have been performed under in vitro condi-tions, using such standardized systems and assays as transepithelial

toxicity or cellular transport and few in vivo reports have been pub-lished of their bioavailability and biocompatibility. Some recentstudies, employing mice as animal models, have focused mainly onestablishing the maximum tolerated doses for PAMAM dendrimers

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f various generations and surface charges, either administeredrally or intravenously. It was observed that when administeredntravenously, the amine-terminated dendrimers G4.0-NH2 and7.0-NH2 were tolerated only at doses not exceeding 10 mg/kg.lso, the lower generations of dendrimers, namely G3.0-NH2 and5.0-NH2, remained non-toxic when given at doses of around

× 10−4 mmol/kg (3.4 and 14.4 mg/kg, respectively). In contrast,arboxyl- (G3.5-COOH and G6.5-COOH) and hydroxyl- (G4.0-OHnd G7.0-OH) terminated dendrimers were safely administeredntravenously at doses 50-times higher or more (Sadekar et al.,011). Sadekar and Ghandehari (2012) have also observed thatAMAM dendrimers show similar toxicities regardless of theiroute of administration, orally or intravenously; although, atigher doses, they were tolerated better when administered orallySadekar and Ghandehari, 2012). Furthermore, the authors demon-trated that the higher generations of cationic dendrimers were, ineneral, more toxic than their lower generation counterparts andhat anionic dendrimers always appeared much less toxic than theationic ones (Sadekar and Ghandehari, 2012).

This paper is a continuation of our earlier reports describinghe impact of 0.5 �mol/kg/day PAMAM G4 on the markers of long-erm metabolic impairments, overall survival in rats suffering fromong-lasting diabetes (Labieniec et al., 2008), as well as their bloodrain-barrier permeability (Karolczak et al., 2012) and mitochon-ria functionality (Labieniec and Gabryelak, 2008; Labieniec andatala, 2010; Siewiera and Labieniec-Watala, 2012).Our earliest report notes that PAMAM G4 mimics the action

f hypoglycaemic agents in reducing plasma hyperglycaemiand long-term markers of poor metabolic control in the animalodel of diabetes: protein glycation and glycoxidation, as well as

ther markers of oxidative and carbonyl stress (Labieniec et al.,008). Nevertheless, despite these apparently mitigating effects oniabetes-associated complications, the overall impact of the den-rimer on animal mortality was negative: such beneficial effectso not outweigh the negative influence of PAMAM’s G4 cytotox-

city and its contribution to lowered overall animal survival. Theutcomes of some other studies support our findings. In theirast study, Dong et al. determined both plasma glucose levels andlasma insulin concentrations in rats administered with PAMAMendrimers and demonstrated that the agents effectively increasedhe pulmonary absorption of insulin without any membrane dam-ge to the respiratory tissues (Dong et al., 2011). This remarkableypoglycaemic effect, seen after pulmonary administration of

nsulin with PAMAM dendrimers, indicates that dendrimers mayossibly be used as carriers in anti-diabetic therapy. These resultsave further stimulated interest in searching for the best non-

nvasive way of dendrimer delivery.In searching for the possible mechanism(s) behind PAMAM

ction, it was proposed that the adverse effects of “PAMAMherapy” may not derive so much from the cyto/toxic action of den-rimers themselves, but rather from the route of the administrationf PAMAM to living organisms. To the best of our knowledge, noublished records based on in vivo studies, conducted on rats with

ong-lasting non-cured diabetes, detailing the impact of differentoutes of PAMAM dendrimer delivery on animal survival currentlyxist. The studies ongoing in our lab are focused on understandinghe in vivo toxicity and bioavailability profile of PAMAM dendrimersnd to finding the safest route of PAMAMs delivery into an organ-sm.

Therefore, the aim of this work was to assess the most effec-ive and the least deleterious route of dendrimer delivery toats with experimental diabetes and chronic hyperglycaemia. To

est the hypothesis that the manner of dendrimer administra-ion may contribute to reduced animal survival, three differentoutes of dendrimer administration were monitored: intraperi-oneal, intragastrical or subcutaneous. Each route of administration

al of Pharmaceutics 464 (2014) 152–167 153

used PAMAM G4 at a dose of 0.5 �mol/kg/day (Labieniec et al.,2008), daily for 60 days, to male Sprague-Dawley adult rats withexperimental diabetes.

For the sake of possible future applications, overall rat survivalwas assessed. In addition, the most important diabetes-associatedindicators of metabolic impairments were recorded in animalswhich received the dendrimer and survived the 60-day experiment.

2. Materials and methods

2.1. Chemicals

PAMAM dendrimer, ethylenediamine core, generation 4.0(PAMAM G4, [NH2(CH2)2NH2]:(G = 4); dendri PAMAM(NH2)64;MW 14,214 Da) (10% (w/w) methanol solution) and streptozotocinwere obtained from Sigma–Aldrich (Germany). Kits for routine bio-chemical determinations were obtained from Roche Diagnostics(Switzerland). Accucheck Active glucose strips were obtained fromRoche Diagnostics Polska Ltd. (Warsaw, Poland). Kits for determi-nation of glycated haemoglobin (HbA1c) were obtained from Drew103 Scientific Ltd. (Barrow-in-Furness, Cumbria, United Kingdom).Ketamine and sedazin for animal anaesthesia were purchased fromBiowet-Pulawy (Poland). All other reagents and solvents used inthis study were of the highest analytical reagent grade and werepurchased from Polish Chemicals (POCH, Gliwice, Poland).

2.2. Animals and husbandry

Male Sprague-Dawley rats, weighing approximately 240–360 g,were used in this experiment. Before the start of the experiment,the animals were acclimatized to the laboratory conditions for aperiod of 2 weeks. They were maintained at an ambient temper-ature of 25 ◦C and a 12/12 h light/dark cycle. Animals were givenstandard commercial rat chow and water ad libitum and werehoused under standard environmental conditions until treatmentor sacrifice. The experiments were conducted in accordance withthe Guide for the Care and Use of Laboratory Animals publishedby the US National Institute of Health (NIH Publication No. 85-23, revised 1985), as well as with the guidelines formulated bythe European Community for the Use of Experimental Animals(L358-86/609/EEC) and the Guiding Principles in the Use of Ani-mals in Toxicology (1989). All experiments were carried out withthe approval of an appropriate institutional local ethics committee.

2.3. Induction of streptozotocin diabetes in rats

Diabetes was induced by intraperitoneal injection of streptozo-tocin (STZ, dissolved in 0.1 mol/l citrate buffer, pH 4.5) at a doseof 70 mg/kg b.w. Diagnosis of diabetes was made on the basisof the non-fasting blood glucose concentration (measured in themorning hours, 08:00–10:00 AM). Animals with blood glucose con-centrations higher than 16.7 mmol/l, were considered diabetic andincluded to the study. Each STZ-injected rat showing hypergly-caemia lower than 16.7 mmol/l at 72 h after injection was excludedfrom the study. The evaluation of daily doses of 0.5 �mol/kg b.w.PAMAM G4 began 7 days after the induction of laboratory con-firmed diabetes. At the termination of the experiment, the survivorsin all tested groups were sacrificed and their blood was collectedfor further biochemical analyses.

2.4. Groups and treatment

Experimental animals (n = 200) were allocated to two cohortsusing the scheme of a randomized block design: rats withoutdiabetes (healthy animals) and animals given streptozotocin (dia-betic rats), assuming a diabetic: healthy ratio of 1.5. In both

154 M. Labieniec-Watala et al. / International Journal of Pharmaceutics 464 (2014) 152–167

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Fluorescence intensity was expressed in arbitrary units (a.u.) per gof plasma albumin.

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ig. 1. Scheme of the random blocked in-series and independent-group allocationsroups with various routes of dendrimer delivery. The unequal group sizes originaortality in various subgroups of animals.

roups, animals were given either PAMAM dendrimers or vehi-le, as described in detail below. Next, the animals of each cohortere further randomly allocated into three groups according to the

oute of administration of either PAMAM G4 dendrimer or a vehi-le (dendrimer solvent, methanol): intraperitoneal, subcutaneousr intragastric.

PAMAM G4 stock solution, dissolved in methanol (10% (w/w)olution), was further diluted 15-fold in physiological saline anddministered to animals either by intraperitoneal or subcutaneousnjection or given intragastrically using a gavage. Regardless ofhe chosen administration route, both the aliquots of PAMAMendrimers or a vehicle were given daily for 60 days, i.e. untilhe experiment was terminated. The dose of PAMAM dendrimer0.5 �mol/kg b.w.) was chosen based on earlier studies (Karolczakt al., 2012; Labieniec et al., 2008; Labieniec and Watala, 2010),n which animals received dendrimers by intraperitoneal injec-ion. Administration of a vehicle, pure methanol diluted 15-foldn physiological saline, served as a control.

The minimum sample size in every group was estimated assum-ng the standardized effect of 1.25–1.40 for the leading variable,bA1c, critical significance and statistical power values of 0.05 and.80, respectively, pre-treatment mortality of 10% and case:control,atio of 1.0 (Fig. 1).

.5. Animal survival, body weight and glycaemia levelvaluations

The overall survival of animals was evaluated based on theaplan–Meier curves. Changes in the body weight and blood glu-ose concentration in all tested animal groups during the 60-daybservation period were recorded every week, between 9.00 and0.00 in the morning. The results were expressed as the meanhange in body weight or glucose level ± SD.

.6. Blood collection and biochemical measurements

Animals were anaesthetized with i.m. injection of ketamine · HCl100 mg/kg b.w.) and xylazine (10 mg/kg b.w.). Blood was collected

n-diabetic rats and animals with experimentally induced diabetes to the treatmenttly from not-completely effective streptozotocin induction of diabetes and animal

from the abdominal aorta of anaesthetized rats and immedi-ately subjected to the separation of blood cells from plasma(Dobaczewski et al., 2006). In-life non-fasting blood glucose levels(recorded always between 09:00–10:00 AM) were measured twicein control non-diabetic animals, at the beginning and at the end ofthe experiment, and monitored weekly in all STZ diabetic animalsunder study. Blood was obtained from the tail vessels by needleprick and tested using glucose strips or, when exceeding 600 mg%(33.3 mmol/l), with a biochemical analyzer. The final determina-tion of blood glucose was recorded within the week preceding thecritical event and referred to as terminal glucose.

Routine biochemical determinations, including blood morphol-ogy and serum proteinograms, were performed with the ABXPENTRA 120 DX (Horiba Ltd., Kyoto, Japan) analyzer and theHydrasys LC semianalyser (Sebia, Lisses, France), respectively.

2.7. Glycated haemoglobin (HbA1c) level determination

Glycated haemoglobin (HbA1c) was analyzed using a commer-cial kit (Drew Scientific Ltd., Barrow-in-Furness, Cumbria, UnitedKingdom), measurements were performed with the DS5 instru-ment, and the results expressed as the percentage fraction of totalhaemoglobin.

2.8. AGEs and AOPP determination

The determination of advanced glycation end-products (AGEs)in blood plasma (fluorescent products including pentosidine andcarboxymethyllysine) was based on spectrofluorometric detection.

mined spectrophotometrically (340 nm) by microplate assay withchloramine T and acetic acid. AOPP concentration was expressed inchloramine units (�mol/l). These procedures are described in detailin Kalousova et al. (2002).

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.9. Statistical analysis

All measurements were performed in duplicate or in triplicate:ll data being expressed as mean ± SD or median and interquartileange from the lower 25% quartile to the upper 75% quartile. Dataormality was checked using the Shapiro–Wilk test, and varianceomogeneity was verified with Levene’s test. Following this, data

ound to have a normal distribution was analyzed with parametricests. The statistical significance between homogenous groups wasstimated using either two-way or one-way ANOVA and post hocukey tests. The non-parametric Kruskal–Wallis test, followed byonover–Inman all pairwise comparisons test, were used for theariables showing heterogeneity of variance.

For time-to-event analysis of data, the Kaplan–Meier methodas used to analyze the survival of animals. The event of inter-

st was survival, whereas the explanatory data were the changesn body weight and glycaemia between the initial point, occurringt the start of PAMAM G4/vehicle administration, and the termi-al point, occurring in the final week preceding the critical event.hile in all cases, the starting point was regarded as the date of the

rst administration of the PAMAM G4/vehicle, the ending point wasnimal death for uncensored (complete) data. Censored (incom-lete) data was obtained from animals, which remained alive athe termination of this study or died of causes unrelated to the

reatment of interest. For the purpose of the time-to-event anal-sis three grouping variables were employed: the health statusnon-diabetic vs. diabetic animals), the compound given (a vehicles. PAMAM G4) and the route of administration (intraperitoneals. intragastrical vs. subcutaneous). The log-rank (Peto) test fortratified or combined data was used to compare survival curves.ox proportional hazard regression analysis was used to assesshe associations between explanatory variables (weight drop, gly-aemia gain) and survival rate, with the assumption that a ratio riskreater than 1 or lower than 1 denotes respectively an increased or aecreased) risk for those with a given characteristic. Kaplan–Meierurvival estimates, survival rate and relative hazard, were esti-ated according to Kaplan and Meier (1958) and Peterson (1977),

espectively. Briefly, the survival rate has been expressed as theurvivor function (S): S(t) = (number of individuals surviving longerhan t)/(total number of individuals studied), where t denotes aime period known as the survival time, time to failure or time tovent (such as death) (Kaplan and Meier, 1958). The cumulativeazard function (H) has been estimated as the risk of event (e.g.eath) at time t: H(t) = − ln(S(t)) (Peterson, 1977).

The sample size was estimated for type I and II statistical errorsf 0.05 and 0.8, respectively. The leading variable was the four-arametric comprehensive score of the reductions in the hallmarksf severe hyperglycaemia (see below) and the expected standard-zed effect was assumed equal to at least 1.0. The sample sizestimated for such assumptions was at least 14, or 20 when cor-ected for increased mortality of causes unrelated to the treatmentf interest. Furthermore, the post hoc power of the estimates washecked for each analysis. A statistical power below 80% was con-idered as an invalid outcome and constructive conclusions wereot formulated.

In order to evaluate the effectiveness of the treatment with

Comprehensive Score (CS) = [Std (x

+ [Std (x/SD) : AGEsPAMAM − 9

endrimers, the overall, comprehensive effectiveness of the hypo-lycaemizing effects of PAMAM G4 were monitored using thealculus approach earlier elaborated in our laboratory and reportedn several papers (f.i. Karolczak et al., 2013). Here, four different

al of Pharmaceutics 464 (2014) 152–167 155

parameters characterizing the hypoglycaemizing, anti-glycationand anti-glycoxidation effects of PAMAM dendrimers were used:(a) non-fasting whole blood glycaemia (mmol/l), (b) glycatedhaemoglobin (HbA1c, %), (c) advanced glycation end-products inblood plasma (AGEs, a.u./g plasma albumin), and (d) advanced oxi-dation protein products (AOPP, �mol/l) (Labieniec et al., 2008).Obviously, each of the above parameters is determined using adedicated methodology and is characterized not only by a natu-ral biological variability, but also by the method-derived variabilitywith regard to the response of an organism to the action of admin-istered PAMAM G4 dendrimer. Importantly, individual changes inthe response to PAMAM administration, assessed by each of fourmethods, depend on the biological variability in the metabolism ofindividual animals.

In order to quantify the extent to which PAMAM reduced thehallmarks of hyperglycaemia, multiplicative criteria were used tocalculate the differences between the observed values of glycaemia,HbA1c, AGEs and AOPP and the accepted reference threshold val-ues (mean ± 95%CI) observed in diabetic animals not given thedendrimers (31.64 mmol/l for blood glucose, 8.36% for HbA1c,9.26 a.u./g albumin for AGEs and 12.76 �mol/l for AOPP). Such dif-ferences, standardized to SD values, were further used to calculatethe additive (comprehensive) scores (CS) of the four-parametricresponse of studied animals to the administration of PAMAM den-drimers, using the following formula:

: glycaemiaPAMAM − 31.64] + [Std (x/SD) : HbA1c (PAMAM) − 8.36]

+ [Std (x/SD) : AOPPPAMAM − 12.76],

where [glycaemiaPAMAM − 31.64], [HbA1c (PAMAM) − 8.36],[AGEsPAMAM − 9.26] and [AOPPPAMAM − 12.76] are the calculateddifferences between the observed values and the accepted thresh-old values for the given variables describing the dendrimer-inducedhypoglycaemizing/anti-glycation/anti-glycoxidation effect, andStd (x/SD) denotes the standardization of such differences towithin-group SD. Hence, the calculated CS values represent theoverall weighted four-parametric measures of the response of theanimals to PAMAM administration in such a way that the lowerabsolute CS value reflects the higher effectiveness of the PAMAMdendrimer in reducing hyperglycaemia/glycation/glycoxidation.

The area-under-the-curve method was employed to determinethe integrated measurements of blood glycaemia and body mass inthe course of the 60-day observation. Due to differentiated animalsurvival in particular groups, the recorded values were standard-ized accordingly.

All statistical calculations were performed with the use of STA-TISTICA.PL v.10 (StatSoft), StatsDirect (StatsDirect Limited, v. 2.7.8.)and GraphPad Prism (GraphPad Software Inc., v. 5.03).

3. Results

3.1. Effect of different modes of dendrimer G4 delivery on animalhealth and overall survival

The effects of the administration of either dendrimer G4 or avehicle (methanol) on the survival of non-diabetic and STZ-diabeticanimals are presented in Fig. 2. As shown, the most significantlyreduced survival times in non-diabetic and STZ-diabetic rats wereobserved upon the intraperitoneal injections of methanol and/orPAMAM G4 dendrimer. Slightly reduced survival of animals was

also noted for intragastric gavage, whereas no death events at allwere recorded for subcutaneous injections, either in STZ-diabeticor non-diabetic rats, regardless of whether PAMAM G4 or a vehiclewere administered.

156 M. Labieniec-Watala et al. / International Journal of Pharmaceutics 464 (2014) 152–167

Fig. 2. The Kaplan–Meier curves of cumulative survival in diabetic animals admin-istered with a vehicle or PAMAM dendrimer G4 using various routes of delivery. Thestep function of the estimated cumulative proportions of survived rats are given for(A) STZ-diabetic animals administered with either a vehicle (methanol) or PAMAMG4, and for (B) non-diabetic and STZ-diabetic animals (pooled together) adminis-tered with PAMAM G4, using three various routes of delivery. Complete observationsare marked with circles, triangles or diamonds, respectively for PAMAM G4 givenintraperitoneally, a vehicle given intraperitoneally or PAMAM G4 given intragas-trically; censorships are marked by ‘ + ’. For the remaining variants of delivery (avehicle or PAMAM G4 given subcutaneously, a vehicle given intragastrically) nodeath events were recorded. The significance of differences between survival curves,gt

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vastly increased (OR from 22.0 in CMI to 87.9 in DMS, p < 0.03 or

iven by log-rank (Peto) test, was: (A) p < 0.0005; p < 0.0002 for the �2 test for therend: G4intraperitoneal > vehicleintraperitoneal > G4intragastrical; (B) p < 0.001.

In all the studied animals, both non-diabetic and diabetic ani-als given either a vehicle or PAMAM G4, the Kaplan–Meier

stimates of the 60-day survival rate were 84.4 ± 3.1% for the ani-als with intraperitoneal injection (group 1), 92.5 ± 3.2% for the

ats with intragastrical administration (group 2) and 100% forhose injected subcutaneously (group 3) (p < 0.01 by the general-zed Wilcoxon–Gehan–Breslow test and log-rank test, p < 0.0001or a trend). Mean survival time was 56.7 days (95%CI: 55.2–58.2ays) for the intraperitoneally-injected group and 56.8 days (95%CI:3.8–59.8 days) for the animals with intragastrical administra-ion. Correspondingly, 10-percentile survival time was 42.5 daysor group 1 (intraperitoneal) and over 60 days for group 2 (intra-astrical), which means that the estimated 10% of animals injected

ntraperitoneally with PAMAM G4 or a vehicle will die within 42.5ays after the starting point, while the other 90% will either notie or will experience the event more than 42.5 weeks after the

Fig. 3. Skin irritation and inflammation observed in animals given PAMAM G4 den-drimers injected intraperitoneally. The arrow indicates hair loss and abscesses filledwith sanguine pus.

starting point. In contrast, 10% of the rats given either the dendrimeror a vehicle by intragastric gavage will not die within 60 days, i.e.they will survive for the overall observation period. Cox propor-tional hazard regression analysis, which controlled for the effectsof the body mass reduction and glycaemia gain in the course ofthe observation, indicated that the animals given peritoneal injec-tions were over 3 times more likely to die than those administeredthe dendrimer or a vehicle intragastrically, and over 9 times morelikely than those administered subcutaneously (the hazard or riskratio [HR]intraperitoneal vs. intragastric = 3.02; 95%CI: 1.21–7.57, p < 0.02;HRintraperitoneal vs. subcutaneous = 9.10; 95%CI: 1.47–56.36; p < 0.02).Moreover, the animals given PAMAM G4 were over 12 timesmore likely to die than those given a vehicle (HR = 12.32; 95%CI:1.57–96.92; p < 0.02).

Comparing all the delivery routes, it was found that subcuta-neous injection was by far the least harmful treatment, the use ofintragastric gavage to administer PAMAM G4, but not a vehicle,resulted in single fatal events in both control (non-diabetic) andSTZ-diabetic animals, while the intraperitoneally supplementedgroup was characterized by the lowest survival ratio. In sum-mary, in animals given the dendrimers intraperitoneally, no deathevents were recorded for vehicle and 4 fatal events for PAMAMG4 in non-diabetic rats, while one death was recorded for vehicleand 6 for PAMAM G4 in diabetic rats (non-significant differences).Otherwise, 4 non-diabetic and 1 STZ-diabetic animal died uponintragastrical administration of PAMAM G4, while no fatal eventsoccurred for the rats given a vehicle. No fatal events were recordedin the control or diabetic animals injected subcutaneously witheither a vehicle or dendrimer.

The general health condition of animals subjected to dendrimeror a vehicle administration was also reflected by the skin irritationand inflammation states (Fig. 3). As shown, the rats injected dailyfor 60 days with the tested chemicals often experienced the statesof a painful skin irritation and/or inflammation, including hairloss, reddening of the skin, festering wounds and open woundsand abscesses, often filled with blood and pus. Such dermatolog-ical characteristics occurred not only in diabetic rats, but also inhealthy animals, and were typical not only for dendrimer treat-ment, but occurred also in animals given a vehicle (methanol).For all the treatment groups given the dendrimer (or a vehicle)by injections, the incidences of such dermatological lesions were

less), however, no significant differences between these incidenceswere recorded with regard to animal group (OR = 22.0–40.2 in non-diabetic, p < 0.005 or less vs. OR = 37.0–87.9 in STZ-diabetic, p < 0.03

M. Labieniec-Watala et al. / International Journal of Pharmaceutics 464 (2014) 152–167 157

Fig. 4. Fluctuations in body mass in non-diabetic and diabetic Sprague-Dawley rats administered with PAMAM G4 using various routes of delivery. Data of the curvesrepresent fluctuations of body masses in non-diabetic and diabetic animals, given either a vehicle (methanol) (A) or PAMAM G4 (B), recorded over the period of 60-dayobservation. Data in a box-whiskar plot (C), presented as medians and interquartile ranges (lower 25% quartile to upper 75% quartile; n = 10–21), represent weight gain orweight loss, respectively in non-diabetic or diabetic animals over the period of 60-day observation. Significance of differences between various routes of delivery, calculatedw s test,o minist

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ith two-way nested ANOVA and the post-hoc Tukey-Kramer multiple comparisonf groups C and D stand for non-diabetic and diabetic animals, M and P denote the ado intragastric, intraperitoneal or subcutaneous administration.

r less) or for treatment (OR = 22.0–87.9 for a vehicle, p < 0.03 oress vs. 37.0–52.9 for PAMAM G4, p < 0.0005 or less).

.2. Fluctuations in body mass and blood glycaemia in animalspon different modes of PAMAM G4 delivery

The fluctuations in body mass in non-diabetic and STZ-diabeticnimals, given either a vehicle or PAMAM G4 dendrimer, are pre-ented in Fig. 4. In general, in all the groups of diabetic animalse recorded very significant body mass reductions, regardless ofhether they received a vehicle or the dendrimer and regardless

f the route of dendrimer delivery. Otherwise, regular weight gainsere recorded in all non-diabetic animals (p < 0.0001 for all the

omparisons between non-diabetic and diabetic animals) (Fig. 4C).he dynamics of body mass fluctuations were presented in a tripleay. In Fig. 4A and B we show raw data of body mass, recordedeekly, in all studied groups. In Fig. 4C we show weight losses in

iabetic and weight gains in non-diabetic animals expressed as theifferences between the initial (the first day of observation) andhe terminal readings. Fig. 4A and B shows that body mass fluc-uations were not essentially different among the subgroups of

were: p < 0.0001, CMI vs. CPI; p < 0.0001, DMS vs. DPS. Abbreviations: in the symbolstration of either a vehicle (methanol) or PAMAM G4, whereas G, I and S correspond

non-diabetic rats and among the subgroups of diabetic animals,with one exception, however: the weight gains in non-diabeticanimals given PAMAM G4 via intraperitoneal injections remainedsignificantly lower compared to those administered with the den-drimer using intragastric gavage (Fig. 4B). The same was confirmedwhen the differences recorded between the initial (the 1st day ofobservation) and the terminal readings were estimated (p < 0.0001)(Fig. 4C). Overall, neither a vehicle (methanol) nor the dendrimeraffected body mass, and there were also no effects revealed associ-ated with the route of the agent delivery.

To interpret the fluctuations in blood glycaemia of non-diabeticand diabetic rats in the course of the 60-day observation, the mea-surements of the variable over time were integrated and the datawas expressed as the areas under curves with the integers set fromthe first to the 60th day of the experiment. The results of non-fasting blood glycaemia monitored in diabetic rats throughout thewhole period of the experiment are presented in Fig. 5A. Regu-

larly, significantly higher levels of glucose were observed over the60-day period in diabetic rats in comparison with their healthycounterparts (0.39 vs. 1.7–1.9 mol/l, p < 0.0001). While methanolhad obviously no effect on the glucose level, regardless of its

158 M. Labieniec-Watala et al. / International Journ

Fig. 5. Hallmarks of severe hyperglycaemia in non-diabetic and streptozotocin-diabetic Sprague-Dawley rats administered with PAMAM G4 using various routesof delivery. (A) Non-fasting blood glycaemia in diabetic rats administered witheither a vehicle (methanol) or PAMAM G4, delivered intraperitoneally, intragas-trically or subcuteneously. Integrated measurements over the time of observationare expressed as the areas under curves with the integers set from the first tothe 60th day of the experiment. Data are presented as medians and interquar-tile ranges (lower 25% quartile to upper 75% quartile; n = 10–21). The value ofintegrated AUC measurements for non-diabetic animals under experiment was0.39 (0.38–0.39) mol/l. (B) Glycated haemoglobin (HbA1c), advanced glycation end-products (AGEs) and advanced oxidation protein products (AOPP) in non-diabeticand streptozotocin-diabetic rats injected intraperitoneally with either a vehicle(methanol) or PAMAM G4. Data, presented as means ± SD or medians and interquar-tile ranges (lower 25% quartile to upper 75% quartile; n = 9–21), represent absolutevalues of HbA1c [%], AGEs [a.u./g albumin] and AOPP [�mol/l]. Significance of dif-ferences between groups was estimated with one-way ANOVA or Kruskal–Wallistest and the post hoc parametric Tukey test or non-parametric Conover–Inman test#. (A): #p < 0.0001, DMI vs. DPI; #p < 0.0001, DMS vs. DPS; #p < 0.04, DPG vs. DPI;#p < 0.002, DPG vs. DPS. (B) HbA1c: p < 0.0001, DMI vs. DPI; p < 0.0001, CMI vs. DMI;p < 0.0001, CPI vs. DPI; AGEs: p < 0.005, DMI vs. DPI; p < 0.0001, CMI vs. DMI; AOPP:p < 0.005, CMI vs. DMI. Abbreviations: CMI and CPI, non-diabetic rats administeredintraperitoneally with either methanol or PAMAM G4, resp.; DMS, DPS, DMI and DPI,diabetic animals given methanol or dendrimer via subcutaneous or intraperitonealinjections; DPG, diabetic rats given PAMAM G4 intragastrically.

dniddwseiwaad(

by the dendrimer, while AOPP demonstrated a neutral or neg-

elivery route, the administration of PAMAM G4 dendrimer sig-ificantly reduced the level of glycaemia in diabetic rats, when

njected intraperitoneally or subcutaneously. Noteworthy, even iniabetic animals given the dendrimer, blood glucose still remainedefinitely higher compared to non-diabetic animals. Otherwise,hen the dendrimer was delivered intragastrically to STZ-rats, no

ignificant reduction was seen in blood glucose (Fig. 5A). Inter-stingly, the response of animals to PAMAM G4 with regard tots “normalizing” effect was very variable among the individuals

ith diabetes: some of them remained almost refractory to thection of the dendrimer, while in the others, the reductions almost

chieved normoglycaemia levels (not shown). Neither vehicle norendrimer affected blood glucose fluctuations in non-diabetic ratsnot shown).

al of Pharmaceutics 464 (2014) 152–167

3.3. Changes in glycated haemoglobin, plasma advancedglycation end-products (AGEs) and advanced oxidation proteinproducts (AOPP) in animals given PAMAM G4 using differentmodes of delivery

Fig. 5B demonstrates the comparison of the absolute values ofthe variables describing the effects of diabetic hyperglycaemia inboth non-diabetic and STZ-diabetic animals, given either a vehicleor PAMAM G4 as intraperitoneal injections. The values of glycatedhaemoglobin and AGEs were significantly increased in diabetic ratscompared to non-diabetic counterparts, while for AOPP the abovewas true only for the animals given a vehicle (methanol). Impor-tantly, the use of PAMAM G4 dendrimer resulted in the reductionsof HbA1c, AGEs and – to much lesser extent – glycaemia, while it hadno significant influence on AOPP (Fig. 5B). Overall, while the admin-istration of PAMAM G4 considerably reduced glycaemia, glycatedhaemoglobin and AGEs, AOPP was the most refractory to reduc-tion upon treatment with the dendrimer. Importantly, AOPP alsoremained the least affected variable in the course of severe hyper-glycaemia in chronic diabetes, which resulted mostly from eitherthe huge biological variability or low measurement precision ofAOPP (Fig. 5B).

Fig. 6 shows the comparison between various routes of den-drimer delivery in reducing the markers of severe hyperglycaemiain STZ-diabetic rats. In general, the reductions of hyperglycaemiamarkers in the animals given the dendrimers intraperitoneallyor subcutaneously were the opposite of those recorded for therats given PAMAM G4 via intragastric gavage. In the majority ofcases, both intraperitoneal and subcutaneous injections resultedin vast reductions in the hallmarks of severe hyperglycaemia. Theextents of such reductions were equal for both delivery routes forall parameters but one, glycated haemoglobin, in which subcuta-neous administration appeared over two times more effective. Onthe contrary, intragastrical administration of PAMAM G4 was eitherineffective (glycaemia and glycated haemoglobin) or demonstratedlower effectiveness compared to intraperitoneal or subcutaneousdelivery. The exception was AOPP, where a negligible reductionwas revealed merely for intragastrical administration, while nei-ther intraperitoneal nor subcutaneous injections were effective. Tofurther compare the effectiveness of PAMAM G4 in reducing themetabolic consequences of severe hyperglycaemia using variousroutes of the dendrimer delivery, Cochrane meta-analysis “forestplots” were used to assign the contributions of different metabolicmarkers in the overall “hypoglycaemizing” effect of the dendrimer.

Fig. 7 shows the Cochrane odds ratio plots for glycaemia, gly-cated haemoglobin, AGEs and AOPP as the variables dependenton the administration of PAMAM G4 using various routes of den-drimer delivery. The dependent variable was the administrationof either PAMAM G4 (rank 1) or a vehicle (methanol) (rank 0)and the continuous explanatory variables, describing the selectedblood biochemical parameters of long-term glycaemia control,were dichotomized as described below. The opposed values ofdichotomized continuous variables were from below or equal to[mean – 95%CI] (≤reference threshold, rank 0) and above [mean –95%CI] (>reference threshold, rank 1), while the reference thresh-old values were estimated for the groups of streptozotocin-diabeticanimals administered with a vehicle and not a dendrimer. The 95%confidence interval horizontal line distant from OR = 1.0 (verticalsolid line) denotes the significant effect of PAMAM G4 on a givenhallmark of diabetic hyperglycaemia. For intraperitoneal and sub-cutaneous administration of PAMAM G4, glycated haemoglobin,blood glycaemia and AGEs were affected to the greatest extent

ative effect. Otherwise, PAMAM G4 administered intragastricallyhad no significant influence on glycaemia, glycated haemoglobin,AGEs or AOPP, did not significantly affect the four-parametric

M. Labieniec-Watala et al. / International Journal of Pharmaceutics 464 (2014) 152–167 159

Fig. 6. PAMAM G4 dendrimer-induced reductions in the markers of hyperglycaemia in STZ-diabetic rats administered with PAMAM G4 using various routes ofdelivery. Data, presented as medians and interquartile ranges (lower 25% quartile to upper 75% quartile; n = 5–19), represent standardized reductions in hypergly-caemia/oxidation markers (glycated haemoglobin (HbA1c), advanced glycation end-products (AGEs) and four-parametric comprehensive scores (CS), all expressed inarbitrary units [a.u.]), in animals treated with PAMAM G4 dendrimers administered intraperitoneally, intragastrically or subcutaneously. The reductions of all the param-eters, except those marked with #, were significant (vs. control groups given a vehicle; p < 0.001 or less). Significance of differences between various routes of delivery,calculated with nonparametric Kruskal–Wallis test and the post hoc Conover–Inman test, were: glycaemia, p < 0.04, Mesubcutaneous = Meintraperitoneal > Meintragastrical; HbA1c,p aperiton

p aemos

ctivo

btheoitgtoopgsatatvt(

3P

fe

altered in a diabetic state, did not change upon dendrimeradministration. The effect of the route of administration was not

< 0.0001, Mesubcutaneous > Meintraperitoneal > Meintragastrical; AGEs, Mesubcutaneous = Meintr

< 0.02, Mesubcutaneous > Meintraperitoneal > Meintragastrical. Abbreviations: HbA1c, glycated hcore.

omprehensive score, and had a moderately significant effect onhe pooled odds ratio (Fig. 6). Overall, the analysis shows that bothntraperitoneal and subcutaneous delivery of the dendrimers hadery significant effects, while the intragastrical delivery had noner very subtle “hypoglycaemizing” effects.

While Figs. 6 and 7 indicate correspondence or even equalityetween intraperitoneal and subcutaneous treatment with regardo the effectiveness of PAMAM G4 in reducing the markers of severeyperglycaemia in chronic diabetes, Fig. 8 clearly presents the high-st benefit/cost ratio recorded for the subcutaneous administrationf the dendrimer. This figure presents the data of the reductionsn these hyperglycaemia markers upon their standardization forhe cumulative relative hazard (mortality) observed in animalsiven the agent by various routes of delivery. As mentioned above,he subcutaneous administration of PAMAM G4 was entirely freef adverse events, including fatalities. Correspondingly, this routef dendrimer delivery appeared by far the most effective in sup-ressing elevated glycaemia, and its metabolic hallmarks, includinglycated haemoglobin and AGEs. For the majority of the markers,tandardized effectiveness did not differ between intraperitonealnd intragastric administration: while the former was more effec-ive in reducing the hallmarks of severe hyperglycaemia, it waslso associated with the increased incidence of adverse effects. Inurn, intragastric administration was not so critical for animal sur-ival, however, it also appeared relatively ineffective in suppressinghe metabolic consequences of severe diabetic hyperglycaemiaFig. 8).

.4. Changes in biochemical parameters upon different modes ofAMAM G4 delivery

Selected biochemical parameters monitored in blood obtainedrom non-diabetic and diabetic animals, that survived the 60-dayxperiment upon the administration of either vehicle (methanol)

eal = Meintragastrical; AOPP, p < 0.01, Mesubcutaneous = Meintraperitoneal > Meintragastrical; CS,globin; AGEs, advanced glycation end-products; CS, four-parametric comprehensive

or PAMAM G4, are summarized in Table 1. The majority of param-eters (incl. aminotransferases, lipids or urea and uric acid) werefound to have significantly increased values in diabetic animals.Only the single variables (total protein and albumin concentrations,�1-globulin fraction) were decreased in diabetes. For numerousvariables (lipids, aminotransferases, urea, fraction of albumins) sig-nificant PAMAM G4-modulating effects were seen; the oppositewas seen in those induced by a diabetic state, i.e. the dendrimerreduced the increased values recorded in diabetic rats, often toa level characteristic of healthy non-diabetic animals. The routeof administration was found to be significant for the choles-terol, aminotransferase, urea and albumin fractions, where, ingeneral, intraperitoneal and subcutaneous delivery appeared toremain much more effective than intragastrical administration(Table 1).

3.5. Changes in blood morphology upon different modes ofPAMAM G4 delivery

The blood morphology parameters in non-diabetic and dia-betic rats treated with either PAMAM G4 or with a vehicle areshown in Table 2. The majority of the measured parametersassociated with white blood cells, lymphocytes, granulocytes andblood platelets became reduced upon the induction of diabetes,those concerning red blood cells remained either unchanged oreven slightly elevated in diabetic animals (MCH, MCV). In gen-eral, the majority of blood morphology parameters, although

particularly discriminative, however, the intraperitoneal or sub-cutaneous administration of the dendrimer were found to beassociated with more acute dendrimer-induced changes, if present(Table 2).

160 M. Labieniec-Watala et al. / International Journ

Fig. 7. Cochrane odds ratio plots for the selected hallmarks of severehyperglycaemia in streptozotocin-diabetic Sprague-Dawley rats administeredwith PAMAM G4 using various routes of delivery. Fixed effects Mantel-Haenszel pooled odds ratio and exact Fisher one-sided significance wereOR = 4.67 (95%CI = 1.86–11.72, pF1� < 0.0005) for intraperitoneal injections (A),OR = 3.24 (95%CI = 1.10–9.52, pF1� < 0.03) for intragastric gavage (B) and OR = 3.54(95%CI = 1.48–8.51, pF1� < 0.0005) for subcutaneous injections (C). The sets of data forthe estimation of the pooled OR included blood non-fasting glycaemia, HbA1c, AGEsand AOPP; CS was excluded of this calculus and presented individually. Opposedvalues of dichotomized continuous variables are from below or equal to [mean –95%CI] (≤reference threshold, rank 0) and above [mean – 95%CI] (>reference threshold,rank 1). The reference threshold values for the hallmarks of severe hyperglycaemiawere calculated for the groups of streptozotocin-diabetic animals administered witha vehicle (methanol) and further used in such a way that the greater differencesbetween individual data and [mean – 95%CI] are relevant to the bigger reductions inthe hallmarks of severe hyperglycaemia. The grouping variable was assigned basedon the administration of either PAMAM G4 (rank 1) or a vehicle (rank 0). OR valuespresented on a logarithmic scale; 95% confidence intervals of odds ratios are dis-played as solid squares with horizontal lines (for a given parameter) or a diamond(for all pooled parameters) with a central vertical dotted line denoting the pooledodds ratio itself. The values of individual OR (for given variables included in themodel) are placed on the right in each plot.

al of Pharmaceutics 464 (2014) 152–167

4. Discussion

Undoubtedly, dendrimers have brought tremendous advancesin the field of bio-medicine, and in general, a great optimismis expected regarding their potential applications in biomedicalareas. However, due to the nanometric size of dendrimers, i.e.1–100 nm, it must be borne in mind that they may potentially inter-act effectively and specifically with various cell organelles, suchas plasma membranes, endosomes, mitochondria and the nucleus,and particular components of cells, such as proteins and enzymes.The size of these cellular components, themselves in the nano-metric range (Jain et al., 2010), may influence the non-selectiveuptake of these nanoparticles, contributing to their cytotoxicity.Regardless of the extensive pharmaceutical and biomedical appli-cations of dendrimers, their toxicity associated with terminal –NH2groups and multiple cationic charges advocates the general opinionthat potential clinical applications of these polymers may thus beseriously limited, even if the in vitro data and physico-chemicalcharacteristics of dendrimers would appear very promising. Asthere is currently a huge commercial interest for the use of den-drimers in various pharmaceutical products, great interest existsin evaluating their toxicity towards mammalian cells.

Our recent studies have repeatedly reported that full-generationPAMAM G4 dendrimers appear very efficient in reducing the in vitronon-enzymatic glycosylation of proteins, as well as the long-termindicators of severe hyperglycaemia in chronic experimental dia-betes in rats (Karolczak et al., 2012; Labieniec et al., 2008; Labieniecand Watala, 2009). However, such desirable modulating effects ofthe metabolic impairments in STZ-diabetic rats came at the price ofdistinct overall toxicity (Labieniec et al., 2008). Thus was the “Janusface” of PAMAM G4 revealed: our studies report that the abilityof dendrimers to scavenge excessive blood glucose and reduce thehallmarks of severe hyperglycaemia goes together with the reducedsurvival of the animals given them. Importantly, we were notable to unambiguously attribute the drastically reduced survivalof animals given the dendrimer to any particular aspect of PAMAMtoxicity (Karolczak et al., 2012; Labieniec et al., 2008). However,such an increased mortality of PAMAM G4-treated animals wasaccompanied with distinct skin irritation and inflammation statesand alterations in blood morphology. Furthermore, our resultsregarding dendrimer toxicity were essentially inconsistent withthose reported by other researchers, who claimed no marked cyto-toxic effects of the dendrimers up to the 5th generation used withincertain dose ranges (Malik et al., 2000; Roberts et al., 1996). All theabove facts prompted us to consider another possible mechanismof the ‘putative’ PAMAMs’ toxicity and to repeat the experimentusing various modes of dendrimer delivery.

Our assumption is that dendrimers may not be so toxic per se atthe used doses, but instead, the route of dendrimer delivery maymatter in determining animal mortality. This assumption led usto conduct another experiment, aimed at testing three differentroutes of dendrimer administration to animals with the experi-mental diabetes. The rationale underlying such an idea is based onsome observations that the accessibility of molecules to any tar-get sites may largely depend upon anatomical and physiologicalbarriers for accessibility of molecules to the target site, which inturn, crucially depends on the route of administration. Thus, thesame dendrimer doses may potentially demonstrate differentiatedbiodistributions and pharmacokinetic profiles, and consequentlyalso differentiated therapeutic efficacy and side effects, based onits route of administration (Wijagkanalan et al., 2011).

The present investigation is the first to compare the effects of

three modes of PAMAM G4 delivery on the general dendrimertoxicity and on the selected markers of long-lasting experi-mental diabetes. Healthy Sprague-Dawley rats, as well as ratswith STZ-induced chronic diabetes, were exposed to PAMAM

M. Labieniec-Watala et al. / International Journal of Pharmaceutics 464 (2014) 152–167 161

Fig. 8. PAMAM G4 dendrimer-induced reductions in the markers of hyperglycaemia adjusted to overall mortality in streptozotocin-diabetic rats administered with PAMAMG4 using various routes of delivery. Data, presented as medians and interquartile ranges (lower 25% quartile to upper 75% quartile; n = 5–19), represent standardizedreductions in hyperglycaemia/oxidation markers (glycated haemoglobin (HbA1c), advanced glycation end-products (AGEs) and four-parametric comprehensive scores (CS),all expressed in arbitrary units [a.u.]), upon their adjustment for overall mortality (relative hazard), in diabetic Sprague-Dawley rats administered with PAMAM G4 dendrimersintraperitoneally, intragastrically or subcutaneously. The logarithmic scale of ordinate used to present very large ranges of values (−100 to 1000). Relative hazard estimatedwith Kaplan–Meier method according to Peterson (1977) (for more details see the text). The reductions of all the parameters, expect those marked with #, were significant(vs. control groups given a vehicle; p < 0.001 or less on transformed data). The significances of differences, calculated with nonparametric Kruskal–Wallis test and thep neal = Mp l = Mei

A ts; CS

Gestgaesrwr(7ddmsc

ipsaivitatInmma

ost hoc Conover–Inman test, were: glycaemia, p < 0.001, Mesubcutaneous > Meintraperito

< 0.0001, Mesubcutaneous > Meintragastrical = Meintraperitoneal; AOPP, p < 0.01, Meintragastrica

bbreviations: HbA1c, glycated haemoglobin; AGEs, advanced glycation end-produc

4 at a concentration of 0.5 �mol/kg b.w./day, administeredither orally (intragastrically) or via injection (intraperitoneally orubcutaneously). The results showed that the dendrimers werehe most effective in suppressing the long-term markers of hyper-lycaemia when using either intraperitoneal or subcutaneousdministration, while the intragastrical route appeared the leastffective. On the other hand, however, the mortality of animalsupplemented with dendrimer was the highest in the group ofats administered with PAMAM G4 intraperitoneally, regardless ofhether control non-diabetic or STZ-diabetic rats were treated and

egardless of whether the rats were given PAMAM or a pure vehiclemethanol). At the end of the observation, lasting for 60 days, only6% and 64% rats survived the whole experiment in the group ofiabetic rats treated, respectively, with a vehicle (methanol) andendrimer G4. However, subcutaneous administration of eitherethanol or PAMAM G4 appeared the least harmful: all the animals

urvived the 60-day experiment, although some dermatologicalomplications were recorded in this group of animals.

What is important, both routes of dendrimer administration,ntraperitoneal and subcutaneous, were equally effective in sup-ressing the long-term markers of severe hyperglycaemia, theecond however, appeared much less harmful with regard todverse effects in the treated animals. Otherwise, although thentragastrical way of delivery was moderately harmful and notery often leading to fatal events, it was also completely ineffectiven reducing the markers of severe hyperglycaemia. This observa-ion may support our point that intragastrical administration mayppear the least harmful at the cost of reduced ingestion, poorerranscellular transport and lower absorption in peripheral tissues.t is noteworthy that the alleviating effects of PAMAM G4 concerned

ot only the altered markers of hyperglycaemia in diabetic ani-als, but also other hallmarks of the diabetes-associated impairedetabolism, including serum lipids (cholesterol and triglycerides),

minotransferases, urea and some protein fractions. While such

eintragastrical; HbA1c, p < 0.0001, Mesubcutaneous > Meintraperitoneal > Meintragastrical; AGEs,ntraperitoneal > Mesubcutaneous; CS, p < 0.02, Mesubcutaneous > Meintraperitoneal = Meintragastrical., four-parametric comprehensive score.

alleviating effects of PAMAM dendrimers for hyperglycaemia-related metabolic impairments seem mechanistically more or lessobvious, the ‘therapeutic’ effects of the dendrimers on the otheraffected parameters are not so apparent with regard to the molec-ular mechanisms of their action and further elucidating studieswould certainly be needed.

It is worth noting that our present observations may support thereasoning that not only the dendrimer per se, but also methanol,can have a negative impact on overall animal survival, when it isgiven via intraperitoneal injections. We have to emphasize at thispoint however, that methanol, used in our study as a solvent fordendrimers, was diluted 15-fold with physiological saline beforeadministration. Therefore, attributing the observed high mortalityof animals to methanol toxicity may seem unjustified. Further, thetreatment was not toxic or was much less toxic in the animals giveneither a vehicle or the dendrimer with the use of the intragastri-cal gavage or by subcutaneous injection. This fact further validatesthe reasoning that it may not be the toxicity of either methanol orPAMAM G4 dendrimers that are toxic for animals, but rather thedelivery route of these compounds may underlie the recorded finaloutcomes of animal survival upon dendrimer/methanol adminis-tration.

In vivo toxicity studies are essential to confirm the safety of anysubstance delivery. Few scientists have performed systematic studyon the in vivo toxicity of PAMAM dendrimers. Evidence accumu-lated hitherto regarding the in vivo monitored safety of PAMAMdendrimers is rather confusing and inconsistent. Importantly,researchers focused on the in vivo testing of PAMAM dendrimersin animal models have employed various routes of their admin-istration; however, to the best of our knowledge, no studies have

compared different routes of dendrimer delivery using the sameanimal model, the same dendrimer doses and similar environmen-tal/laboratory conditions. Moreover, the majority of animal studies(mice and rats), carried out in order to evaluate the in vivo toxicity

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Table 1Blood plasma biochemical parameters in healthy non-diabetic and STZ-diabetic rats treated with PAMAM G4 dendrimer–the effect of the route of administration.

Parameter Non-diabetic animals compound delivered Streptozotocin-diabetic animals compound delivered

Vehicle PAMAM G4 Vehicle PAMAM G4

Route of compound administration Route of compound administration

Intraperitoneal(CMI)

Intragastrical(CMG)

Intraperitoneal(CPI)

Intragastrical(CPG)

Intraperitoneal(DMI)

Intragastrical(DMG)

Subcutaneous(DMS)

Intraperitoneal(DPI)

Intragastrical(DPG)

Subcutaneous(DPS)

Total protein [g/L] 64.6 ± 3.2 64.7(62.6;65.6) 63.5 ± 5.2 61.7 ± 2.6 58.3 ± 5.6 57.7 ± 4.2 56.8 ± 4.5 54.7 ± 6.1 59.2(57.5;62.1) 51.1 ± 4.3Albumin [g/L] 27.8(26.6;28.5) 28.9 ± 1.3 27.7 ± 2.6 27.9 ± 1.2 24.0 ± 2.7 25.8 ± 1.7 26.0 ± 2.5 23.2 ± 3.8 24.9 ± 1.3 20.9(19.4; 21.6)Proteinogram

Albumins [%] 40.9 ± 2.5 42.4 ± 2.6 45.1 ± 2.9 42.6 ± 1.8 41.5 ± 5.5 44.6 ± 2.9 42.9 ± 4.0 38.7 ± 7.5 41.4 ± 3.6 29.6(28.4;34.6)�1-Globulins [%] 26.8 ± 1.8 27.5 ± 1.8 22.3 ± 3.0 26.0 ± 2.0 15.1 ± 4.1 13.1 ± 3.2 14.8 ± 4.8 17.2 ± 4.4 12.3(11.0;15.1) 26.8(24.3;27.2)�2-Globulins [%] 2.8 ± 0.3 2.9 ± 0.3 3.0 ± 0.3 3.0 (2.8; 3.5) 8.7 (6.3; 9.5) 8.7 ± 2.1 7.7 ± 3.3 6.8 ± 2.9 8.2 ± 1.9 4.2 (3.8; 4.8)�-Globulins [%] 23.7 ± 2.5 22.1 ± 1.8 22.8 ± 2.1 21.9 ± 1.7 29.2 ± 5.2 26.0 ± 2.3 27.6 ± 3.0 29.9 ± 4.4 29.5 ± 3.5 32.6 ± 3.5�-Globulins [%] 6.3 ± 1.0 4.9 ± 0.8 6.4 ± 2.3 5.6 ± 0.5 7.4 ± 1.2 6.9 ± 1.3 6.9 ± 1.9 6.8 ± 2.2 7.4 ± 1.5 5.8 ± 1.6

Ala aminotransferase [IU/L] 58 ± 11 64 ± 11 59 ± 11 63 ± 16 171 ± 53 233 ± 87 n/d 108 ± 55 206 (138; 381) n/dAsp aminotransferase [IU/L] 110 ± 22 128 ± 21 129 (121; 144) 111 ± 15 145 (117; 174) 295 ± 160 n/d 114 (102; 172) 171 (137; 354) n/dUrea [mmol/l] 7.6 (7.0; 7.9) 7.9 ± 0.6 9.3 ± 1.7 7.5 ± 0.5 15.8 ± 5.9 11.7 ± 2.5 n/d 11.9 ± 1.8 13.0 ± 2.8 n/dUric acid [�mol/l] 52 (47; 74) 73 (65; 101) 84 (67; 99) 67 (56; 96) 127 ± 62 127 ± 86 n/d 160 ± 98 121 ± 79 n/dTotal cholesterol [mmol/l] 1.44 ± 0.18 1.60 ± 0.16 1.52 ± 0.18 1.40 ± 0.10 1.77 ± 0.38 2.11 ± 0.49 2.02 ± 0.46 1.38 ± 0.30 1.80 ± 0.28 1.37 (1.23;1.44)Triglycerides [mmol/l] 0.86 ± 0.24 0.83 ± 0.31 1.16 ± 0.48 0.76 ± 0.29 1.62

(1.25;3.16)3.19 ± 1.61 1.98 ± 0.81 1.74 ± 1.18 2.02 ± 0.85 1.19 (0.82;1.64)

All parameters in healthy and diabetic rats measured in plasma derived from blood withdrawn from animals that survived the observation. Data are expressed as mean ± SD or median and interquartile range (lower 25% toupper 75% quartile; for variables departing from normal distribution), n = 9–21 animals. Significance of differences estimated with the two-way hierarchical (nested, incomplete) ANOVA on raw or transformed data. Within-groupcomparisons were analyzed with the post hoc multiple comparison LSD Fisher’s test for the nested effects: route (nested in control vs. diabetic) for the vehicle-treated animals and route (nested in vehicle vs. PAMAM) for controlor diabetic animals.Significance level for tested parameters:Total protein: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, n.s.; route, n.s.Albumin: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, n.s.; route, n.s.Proteinogram [% fractions]: albumins: control vs. diabetic, n.s.; vehicle vs. PAMAM, p < 0.002 in control, p < 0.05 in diabetic; route, p < 0.02 in control, n.s. in diabetic.�1-Globulins: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, p < 0.0001 in control and n.s. in diabetes; route, p < 0.0005 in control and p < 0.03 in diabetes.�2-Globulins: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, p < 0.02 in control and n.s. in diabetes; route, n.s.�-Globulins: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, n.s.; route, n.s.�-Globulins: control vs. diabetic, p < 0.02; vehicle vs. PAMAM, p < 0.05 in control and n.s. in diabetes; route, p < 0.01 in control and n.s. in diabetes.Ala aminotransferase: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, n.s.; route, n.s. in control and p < 0.001 in diabetes.Asp aminotransferase: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, n.s.; route, p < 0.001 in control and p < 0.01 in diabetes.Urea: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, p < 0.001 in control and n.s. in diabetes; route, p < 0.0001 in control and p < 0.03 in diabetes.Uric acid: control vs. diabetic, p < 0.001; vehicle vs. PAMAM, n.s.; route, n.s.Total cholesterol: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, n.s. in control and p < 0.005 in diabetes; route, p < 0.002 in control and p < 0.003 in diabetes.Triglicerydes: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, n.s. in control and p < 0.05 in diabetes; route, p < 0.005 in control and n.s. in diabetes.Abbreviations: CMG, control animals given methanol (vehicle) intragastrically; CMI, control animals given methanol (vehicle) intraperitoneally; CPG, control animals given PAMAM intragastrically; CPI, control animals givenPAMAM intraperitoneally; DMG, diabetic animals given methanol intragastrically; DMI, diabetic animals given methanol intraperitoneally; DMS, diabetic animals given methanol subcutaneously; DPG, diabetic animals givenPAMAM Intragastrically; DPI, diabetic animals given PAMAM intraperitoneally; DPS, diabetic animals given PAMAM subcutaneously.

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Table 2Blood morphology in non-diabetic and STZ-diabetic rats administered with PAMAM G4 using various routes of delivery.

Parameter Non-diabetic animals compound delivered Streptozotocin-diabetic animals compound delivered

Vehicle PAMAM G4 Vehicle PAMAM G4

Route of compound administration Route of compound administration

Intraperitoneal(CMI)

Intragastrical(CMG)

Intraperitoneal(CPI)

Intragastrical(CPG)

Intraperitoneal(DMI)

Intragastrical(DMG)

Subcutaneous(DMS)

Intraperitoneal(DPI)

Intragastrical(DPG)

Subcutaneous(DPS)

WBC [103/�l] 8.2 (7.2; 8.9) 5.8 (4.6; 8.7) 6.3 (5.5; 8.5) 5.9 ± 2.3 4.1 ± 2.5 3.7 ± 2.1 5.0 ± 2.0 3.7 (3.1; 6.4) 3.2 (2.6; 4.0) 4.9 (4.2; 5.4)RBC [106/�l] 8.0 ± 0.8 7.7 (6.9; 8.1) 6.9 ± 0.6 7.9 ± 0.7 7.6 ± 1.5 7.8 ± 0.5 8.3 ± 0.6 7.2 ± 1.1 7.1 (6.4; 7.5) 7.3 ± 0.6PLT [103/�l] 723 ± 112 732 ± 54 733 ± 188 664 ± 98 434 ± 180 333 ± 186 489 ± 201 469 ± 277 294 ± 186 787 (501; 900)MCV [fl] 50.2 ± 1.4 50.3 ± 1.7 47.2 (46.7;47.78) 50.0 ± 2.1 55.0 ± 5.7 55.9 ± 2.8 57.7 ± 2.6 52.1 ± 4.6 54.9 (53.8;56.5) 54.0 (51.0;54.0)MCH [pg] 17.3 ± 0.5 17.2 ± 0.2 16.1 ± 0.7 17.0 ± 0.6 18.3 (17.9;18.9) 18.5 ± 0.8 18.4 ± 0.8 16.8 ± 0.3 18.1 ± 0.7 16.3 ± 0.9MCHC [g/dl] 34.5 ± 0.5 34.4 ± 0.4 33.8 ± 0.4 34.1 ± 0.9 33.0 ± 1.1 33.2 (32.8;33.8) 32.1 ± 1.0 32.3 ± 1.0 32.8 ± 0.7 30.8 ± 0.8MPV [fl] 7.4 (7.3; 7.7) 7.2 ± 0.3 7.4 ± 0.6 7.7 ± 0.5 8.5 ± 0.2 8.4 ± 0.4 n/d 8.6 ± 1.1 8.7 ± 0.8 n/dPDW [a.u.] 15.3 ± 0.6 15.5 ± 0.4 14.3 (14.2;14.8) 15.4 ± 0.7 16.0 ± 0.7 15.7 ± 0.9 n/d 15.4 ± 0.9 16.1 ± 0.8 n/dPCT [%] 0.53 (0.47;0.60) 0.52 ± 0.03 0.50 ± 0.12 0.51 ± 0.09 0.33 ± 0.15 0.27 ± 0.14 n/d 0.37 ± 0.20 0.25 ± 0.14 n/dLYMPH [%] 47.0 ± 5.5 42.8 ± 6.5 34.3 (33.0;37.7) 38.7 ± 13.3 33.5 ± 16.1 31.3 ± 15.1 n/d 28.6 ± 11.2 30.0 (21.1;38.3) n/dGRAN [%] 21.9 ± 6.7 16.5 ± 2.5 35.4 ± 7.8 16.6 ± 7.9 13.6 ± 7.9 15.8 ± 11.5 n/d 34.0 ± 19.5 11.7 ± 7.3 n/dMID [%] 31.2 ± 6.8 38.7 ± 3.7 22.9 ± 9.4 38.6

(30.8;59.6)50.9 ± 19.7 50.3 ± 23.4 n/d 38.9 ± 18.3 57.7 ± 16.1 n/d

All parameters in healthy and diabetic rats measured in plasma derived from blood withdrawn from animals that survived the observation. Data are expressed as mean ± SD or median and interquartile range (lower 25% to upper75% quartile), n = 9–21 animals. Significance of differences estimated with the two-way hierarchical (nested, incomplete) ANOVA on raw or transformed data. Within-group comparisons were analyzed with the post hoc multiplecomparison LSD Fisher’s test for the nested effects: route (control vs. diabetic) for the vehicle-treated animals and route (vehicle vs. PAMAM) for control or diabetic animals.Significance levels for tested parameters:WBC: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, n.s.; route, n.s.RBC: control vs. diabetic, n.s.; vehicle vs. PAMAM, n.s. in control and p < 0.0001 in diabetic; route, p < 0.01 in control and n.s. in diabetic.PLT: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, n.s.; route, n.s.MCV: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, p < 0.02 in control and p < 0.001 in diabetic; route, p < 0.04 in control and n.s. in diabetic.MCH: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, p < 0.0002 in control and p < 0.0001 in diabetic; route, p < 0.01 in control and p < 0.02 in diabetic.MCHC: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, p < 0.02 in control and p < 0.0002 in diabetic; route, n.s.MPV: control vs. diabetic, p < 0.0002; vehicle vs. PAMAM, n.s.; route, n.s.PDW: control vs. diabetic, p < 0.03; vehicle vs. PAMAM, n.s.; route, n.s. in control and p < 0.05 in diabetic.PCT: control vs. diabetic, p < 0.0001; vehicle vs. PAMAM, n.s.; route, n.s.LYMPH: control vs. diabetic, p = 0.0005; vehicle vs. PAMAM, p < 0.01 in control and n.s. in diabetic; route, n.s.GRAN: control vs. diabetic, p = 0.06; vehicle vs. PAMAM, p < 0.003 in control and p < 0.02 in diabetic; route, p < 0.003 in control and p < 0.001 in diabetic.MID: control vs. diabetic, p < 0.002; vehicle vs. PAMAM, n.s.; route, n.s.Abbreviations: CMG, control animals given methanol (vehicle) intragastrically; CMI, control animals given methanol (vehicle) intraperitoneally; CPG, control animals given PAMAM intragastrically; CPI, control animals givenPAMAM intraperitoneally; DMG, diabetic animals given methanol intragastrically; DMI, diabetic animals given methanol intraperitoneally; DMS, diabetic animals given methanol subcutaneously; DPG, diabetic animals givenPAMAM Intragastrically; DPI, diabetic animals given PAMAM intraperitoneally; DPS, diabetic animals given PAMAM subcutaneously; RBC, red blood cell count; WBC, leucocyte count; PLT, platelet count; MCV, mean (red blood)cell volume; MCH, mean concentration of haemoglobin; MCHC, mean cell haemoglobin content; MPV, mean platelet volume; PCT, plateletcrit; PDW, platelet distribution width; LYMPH, lymphocyte fraction; GRAN, granulocytefraction; n/d, not determined.

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rofile of dendrimers, has focused on the overall animal survivalates, body weight changes, level of appetite and other behaviouralactors. In contrast, our studies were aimed not only at the mon-toring of animal survival upon exposure to dendrimers, but alsot selecting the most effective and the least harmful method ofendrimer administration.

In brief, Roberts et al., who studied in vivo toxicity of PAMAM3, G5 and G7 in Swiss–Webster mice, observed that only PAMAM7 may produce potential biological complications, and concluded

hat such characteristics of PAMAM dendrimers do not precludeheir use in biological applications (Roberts et al., 1996). Likewise,ajananthanan et al., who studied the immunopotential propertiesf novel formulations including PAMAM G5, found the formulationson-toxic to mice and effective in producing an antigen-specificntibody response (Rajananthanan et al., 1999). In their in vivoiodistribution study, Malik et al. observed that the 125I-labelledationic PAMAM dendrimers administered intraperitoneally orntravenously to Wistar rats were rapidly cleared from circulation,

hile only 0.1–1.0% of the dose was recovered in blood 1 hr afterdministration (Malik et al., 2000). Another detailed study on then vivo acute toxicological profile of melamine dendrimers (2.5, 10,0 and 160 mg/kg), administered to mice by intraperitoneal injec-ion, demonstrated that merely the highest dose resulted in 100%

ortality within 6–12 h post injection. The hallmarks of hepatotox-city, increased liver enzyme activities, occurred at 40 mg/kg, andenal damage was observed above 10 mg/kg, while neither harm-ul effects nor mortality were recorded at the subchronic doses of.5 and 10 mg/kg. From the aforesaid account it may be concludedhat the administration of dendrimers to a biological system needs

detailed study on their in vivo disposition, which depends on theirore structural components, interior generations and particularly,heir surface groups (Neerman et al., 2004). The excellent review,oncerning the pros and cons of various ways of dendrimers’ deliv-ry (including PAMAMs), was presented by Cheng and co-workersn 2008. The authors discussed in detail the studies on variouspplications of dendrimers, with much emphasis paid to differen-iated routes of dendrimer administration, including intravenous,ntraperitoneal, oral, transdermal and ocular administration sys-ems. It has been briefly documented that unmodified cationicendrimers can be administered using different ways of delivery,ut because of their rapid body distribution (to liver, spleen andidney) and potential toxicity, their applications in clinical trailsay become greatly limited. Preferably, the polycationic species

f these nanomolecules may be modified at their surface by var-ous chemical groups in order to minimize their toxicity, often athe cost however, of their attractive electrophilic properties. There-ore, the increasing interest has been paid to the applications of

odified PAMAM dendrimers and different ways of their adminis-ration have been also studied and reported. The results reportedy some researchers have clearly shown that following such sur-ace group modifications, as well as the careful dose selection, theefinement of dendrimers’ molecular geometry and the control-ing of environmental conditions, the modified cationic PAMAMsould be successfully used in various medical applications usingifferentiated ways of drug delivery (Cheng et al., 2008).

Until now, there have been only very few reports on the in vivose of dendrimers as potential drugs per se. Much more often,esearch has focused on the aspects of using dendrimers as drugelivery systems, including drug solubilizing agents, absorptionnhancers, release modifiers and carriers for drug targeting. Poly-eric “drug” delivery systems often fail when applied in oral drug

dministration, as these systems are generally too large for effi-

ient transport across the epithelial barrier of the intestinal tract.owever, the studies of Sweet et al. indicate that dendrimers of a

pecified size and charge can effectively move across gastrointesti-al epithelia (Sweet et al., 2009). Borowska et al. in the in vivo study

al of Pharmaceutics 464 (2014) 152–167

have revealed that the depth of 8-methoxypsoralen (8-MOP) pen-etration and its attained concentration becomes enhanced when8-MOP is encapsulated in PAMAM G3 and G4 (Borowska et al.,2012). These observations indicate that both dendrimer gener-ations are able to penetrate even to deep layers of the skin,although the exact mechanism by which PAMAM dendrimersimprove transdermal drug delivery has not been clarified. Thereare several possible mechanisms underlying the increased penetra-tion of drugs across cellular and biological membranes by PAMAMdendrimers. It has been well evidenced that the large number ofamine groups on the surface of cationic dendrimers could alter theskin barrier function and can potentially facilitate the transport ofdrug–PAMAM dendrimer complexes through the skin (Chauhanet al., 2003). Most relevant studies focusing on the interactionsbetween dendrimers and biological membranes have shown thatas a consequence of the impact of dendrimers on membrane pro-teins, the architecture and composition of the protein–lipid bilayer,cellular lipids or membrane ionic milieu, dendrimers can facilitategreater cell penetration for drugs and increase their aqueous per-meability (Borowska et al., 2012; Cheng et al., 2007; Shcharbin et al.,2006). These findings clearly indicate that PAMAM dendrimers areable to cross biological membranes and reach deeper-located cells,tissues and organs. From this point of view, it is very important torecognize how to use dendrimers at a safe dose and how to choosetheir generations low enough in order to avoid their toxic effects.The dendrimers’ toxicity was found to be concentration/dose-,generation-, as well as time-dependent (Sadekar and Ghandehari,2012). Considering different properties and ambiguous biologicalactivity of PAMAMs, the development of a convenient manner ofthe appropriate selection of their generation, dose, way of deliveryand/or incubation time, along with biodistribution and pharma-cokinetic studies, would be of great interest. It has been wellevidenced that the lower dendrimer generation (below G6), theshorter blood circulation time and the faster accumulation in majororgans. Therefore, it is important to remember that even low gen-erations of dendrimers, used at higher (single or cumulative) dosesor/and administered for longer times of treatment, may have a toxicimpact on the functionality of major organs and may easily lead tovarious systemic dysfunctions (Lesniak et al., 2013).

In general, PAMAM dendrimers have already successfullyproved themselves as useful additives in different routes ofdrug administration, including intravenous, oral, transdermal,and ocular delivery systems, however, the array of accumulatedevidence concerning the safety aspects of various administra-tion routes remains inconsistent (Asthana et al., 2005; Chauhanet al., 2003; D’Emanuele et al., 2004; Wiwattanapatapee et al.,2000). Of these, the pulmonary route has also been suggestedas a useful administration mode of gene delivery with PAMAMdendrimers (Rosenecker et al., 2003). Dong et al. first revealedthe absorption-enhancing activity of PAMAM G2 and G3 den-drimers on the pulmonary absorption of insulin in male Wistarrats. Moreover, insulin complexed with tested dendrimers hasbeen claimed to have a remarkable hypoglycaemic effect afterpulmonary administration. This absorption-enhancing effect wasconcentration dependent, and maximal dendrimer activity wasobserved in the presence of high concentrations (1%, w/v) of eachdendrimer. Importantly, PAMAM dendrimers appeared safe afterpulmonary administration and caused no damage to lung tissues(Dong et al., 2011). Earlier, Navarro and de Ilarduya showed thatPAMAM dendrimers (G4 and G5), injected intravenously to femalebalb/c mice, are able to generate nanosized particles with plas-mid DNA. These carriers are characterized by low toxicity and

good transfection performance in both in vitro and in vivo experi-ments (Navarro and de Ilarduya, 2009). Also, hydroxyl-terminatedPAMAM dendrimers (G5-OH, G6-OH and G7-OH) have shownpromise as drug carriers for targeted delivery to solid tumours.

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umour-bearing female nude/nude mice were dosed intravenouslyy tail vein injection with dendrimers. The biodistribution resultshowed that, due to selective kidney accumulation of PAMAM G5,he pharmaceuticals should be used with caution in delivery sys-ems of drugs that cause kidney toxicity. Otherwise, dendrimers G6nd G7, which also have the potential to accumulate in the liver,ave not been observed to affect liver function (Sadekar et al., 2011).

The above reports, supporting the idea that the intravenousoute of dendrimer treatment can be safe for both mice and rats,re inconsistent with other reports on the haematological tox-city of cationic dendrimers injected intravenously (Malik et al.,000). With regard to intraperitoneal administration of PAMAMendrimers, the results are even more ambiguous. The studies con-ucted by Roberts et al., as mentioned above, report that cationicendrimers (G3, G5 and G7), when delivered intraperitoneally atoses ranging from 0.026 to 45 mg/kg b.w. and given as a singleose or once a week for 10 weeks, were, more or less, safe for thereated mice, based on observations that no significant behaviouralbnormalities or body weight losses occurred through the study,nd only one, out of five mice used in the experiments, had died4 h after G7 dendrimer injection at the highest dose of 45 mg/kgRoberts et al., 1996). On the other hand, Heiden et al. showed thatAMAM G4, when used at 20 �M, was toxic to zebra fish embryos.

mortality of 100% was observed 24 h after fertilization in thesh given the dendrimer. By contrast, in the same study anionicAMAM dendrimers (G3.5) were not toxic for embryo developmentHeiden et al., 2007). Lastly, in rabbits injected intraperitoneally for0 days with PAMAM G3, G5 and G7 (at a dose of 5 × 10−5 mmol)o evidence of immunogenicity was found (Roberts et al., 1996).

Considering the above reports, it seems that regardless of den-rimer delivery route, the in vivo toxicity or increased animalortality after dendrimer administration, depends rather on the

sed dose, dendrimer generation or treatment duration, whilehere is no information from other investigators on the possiblempact of the mode of PAMAM administration on dendrimer toxic-ty. However, earlier studies concerning the use of PAMAMs were

ore concerned with the desirable, beneficial consequences of these of dendrimers as delivery systems, and not so much with theafety aspects of using PAMAM dendrimers as the curing agentser se. This paper emphasizes the harmful effects of intraperitonealelivery of PAMAM G4, and these are compared with the effects ofwo other delivery modes: intragastric gavage and subcutaneousnjection. After an experiment lasting for 60 days, intraperitonealnjection of PAMAM G4 was seen to be by far the most harmful

ode of delivery, causing nearly 20% and over 35% mortality in non-iabetic and STZ-diabetic animals. Intragastrical gavage appeareduch less harmful, while subcutaneous injections remained com-

letely harmless with regard to animal mortality.Overall, these results indicate that various factors play a role in

etermining the overall survival of animals treated with PAMAM4. First, intraperitoneal administration is more harmful than othermployed routes of administration. Second, the injections withAMAM G4 seem more harmful than administration with a vehi-le (methanol). Third, the animals with metabolic impairments,xperimental diabetes, were more prone to die in the course ofhe treatment than non-diabetic rats. Altogether, it is likely thathese three factors, the dendrimers toxicity, the route of dendrimerdministration, and lastly, the health status, may coincide to resultn increased mortality.

Taking into account our present results, as well as our previ-usly acquired data (Karolczak et al., 2012; Labieniec-Watala andiewiera, 2013; Labieniec et al., 2008; Siewiera and Labieniec-

atala, 2012) it appears that at least two other confounding vari-

bles may have a considerable impact on the estimated overallnimal survival in the course of dendrimer ‘therapy’: the sig-ificance of the animal strain (Wistar vs. Sprague-Dawley rats)

al of Pharmaceutics 464 (2014) 152–167 165

used in a study and the effect of seasonality (spring–summervs. autumn–winter period) on the acquired data. Throughout ouryears of experience with the rat model of streptozotocin-induceddiabetes, we have observed that Wistar rats are not only moresensitive to the induction of experimental diabetes with strepto-zotocin, but they also demonstrate more profound discriminationboth in various biochemical markers and overall mortality betweendendrimer- or placebo-treated animals (Labieniec-Watala et al.,2011), compared to Sprague-Dawley rats (Karolczak et al., 2012;Siewiera and Labieniec-Watala, 2012).

When considering other papers using the animal (rat) model ofdiabetes, it appears that Wistar rats (Ates et al., 2007; Khaliq et al.,2013; Moree et al., 2013; Ugochukwu and Cobourne, 2003) are usedwith approximately the same frequently as Sprague-Dawley rats(Ihm et al., 1999; Posuwan et al., 2013; Przygodzki et al., 2011).However, no available reports purposefully compare the sensitiv-ity of these two rat strains with regard to animal susceptibilityfor developing experimental diabetes, as well as animal sensitivityto a given pharmacological treatment or animal mortality. There-fore, at present it is impossible to do anything more than speculateon the differentiated mortality, susceptibility or sensitivity of ratsoriginating from different strains.

Another aspect worth mentioning concerns the differencesin the response to treatments that are dependent on seasonalfluctuations. Our earlier study on dendrimers demonstrates thatthe biological and pharmacological characteristics of biologicalmaterial derived from animals during autumn or winter may sig-nificantly differ from those taken in spring or summer (Siewieraand Labieniec-Watala, 2012). Seasonal variations influence themetabolism of laboratory animals and were found to significantlyaffect study outcomes.

The data demonstrating that laboratory animals (rats) may showcircannual variations, consequently reflected in the values of mon-itored parameters, is continually growing (Bhat et al., 2008; Diazet al., 2011; Konior et al., 2011). Intriguingly, the effects of sea-sons on organism biochemistry and metabolism have also beenseen in humans, which adds further weight to the phenomenonof seasonality. Brachial artery flow mediated dilation, monitoredas a measure of endothelial function, has been shown to reach thehighest values in summer and the lowest in winter (Widlanskyet al., 2007). Elegant evidence from studies on guinea pigs, Wistarrats and human volunteers, validating the significance of seasona-lity, suggests that the seasonal oxidative stress may be a commonphenomenon and may both directly and indirectly affect the func-tionality of a plethora of proteins, including enzymes, as well asorgans, such as the heart. This interesting and important studyclearly demonstrates that the overproduction of oxidative stressand downregulation of numerous peroxide metabolism enzymeswas typical for late spring and summer period, while it was non-existent in other parts of the year (Konior et al., 2011). In general,the overall array of data concerning the effects of seasonality onvarious aspects of metabolism under study remains inconsistent inconfirming that either the spring/summer or autumn/winter sea-sons are more sensitizing to tested agents (Bhat et al., 2008; Michelet al., 2011; Workman and Nelson, 2011).

In summary, the subcutaneous administration of PAMAM den-drimers represents the best compromise between the adverseenhanced toxicity of dendrimer administration and anti-glycationactivity of the dendrimers. For this reason, this route of dendrimerdelivery definitely appears the optimal choice, at least in the caseof PAMAM G4. The question of whether the same rationale remainsvalid for other generations of PAMAM dendrimers is open for futureinvestigations. Thus, when considering the use of PAMAM den-drimers as pro-pharmaceutics in the treatment of hyperglycaemia

in diabetes, we do not need to be forced to choose between ‘thelesser of two evils’.

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. Conclusions

The current findings suggest that numerous metabolic impair-ents of long-lasting and untreated experimental STZ-induced

iabetes in rats can be effectively reduced and controlled via thentraperitoneal or subcutaneous injection of PAMAM dendrimers.ur previous studies have unambiguously demonstrated that

he most hyperglycaemia markers become drastically suppressedsing PAMAM G4, although–at the same time, the dendrimer

argely increases the mortality of diabetic animals when admin-stered intraperitoneally. Our present study expands the positiveutcome of earlier studies by showing that the alternate routef dendrimer delivery, subcutaneous injection, effectively takesway the burden or threat of intraperitoneal administration,hile still maintaining the advantageous aspect of the treat-ent. Accordingly, the present data encourages the idea that

ong-term treatment with PAMAM dendrimer may be potentiallyurable towards limiting the development of diabetic complica-ions dependent on severe hyperglycaemia, however, considerablettention has to be paid to choosing the appropriate route of deliv-ry and pharmacokinetic profile of the agent. Noteworthy, theotentially beneficial effects of dendrimer administration do noteed to be restricted merely to the long-term markers of hyper-lycaemia – they may expand far beyond the impairments ofarbohydrate metabolism, although the mechanisms of such activ-ties may remain elusive at present.

Overall, we hypothesize that PAMAM dendrimers may serve asn alternative therapeutic agent for treatment of a burden of dia-etes mellitus, however, further studies are required in order torecisely determine the conditions of such treatment.

onflicts of interest statement

The authors declare that there are no conflicts of interest.

cknowledgements

This work was supported by the grant from the Ministry of Sci-nce and Higher Education, no. N N405 261037. Authors thankatarzyna Maczynska, Kamil Karolczak and Bartosz Grobelski for

heir excellent technical support.

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