Characteristics of the polymers
The PAMAM-OH dendrimers under study were generations 5.0, 6.0 and 7.0 with hydroxyl surface groups. These generations were chosen such that their molecular weights (29, 58 and 117 kDa) lie in the physiologically relevant range for kidney filtration, extended plasma circulation and tumor retention. The two HPMA copolymers synthesized (26 and 52 kDa) were fractionated in order to have comparable molecular weights with PAMAM G5.0-OH and G6.0-OH. The HPMA copolymers were synthesized with 20 mole percent glycine-glycine-ethanolamine side chains in order to provide the linear polymer backbone with pendant groups that typically facilitate the attachment of bioactive and imaging agents4, 20
. The polymeric side chains terminated in hydroxyl groups, similar to the terminal groups of PAMAM-OH dendrimers under study in order to minimize the influence of surface or pendant functional group characteristics on comparative biodistribution.
Both the PAMAM-OH dendrimers and HPMA copolymers eluted in the order of decreasing molecular weight within the same polymer series. Comparing the elution times of HPMA copolymers and PAMAM-OH dendrimers of comparable molecular weights, the HPMA copolymer of 52 kDa eluted slightly before the PAMAM dendrimer G6.0-OH (58 kDa) (). The hydrodynamic radius of the HPMA copolymer of 52 kDa was also higher than that of PAMAM G6.0-OH () as determined by dynamic light scattering. However, in case of the lower molecular weight HPMA copolymer of 26 kDa, an opposite trend was observed with this copolymer eluting after the PAMAM dendrimer G5.0-OH (). The hydrodynamic radius obtained by dynamic light scattering was also consistent with the observed trend, with a radius of 1.4 nm for the HPMA copolymer and 2.3 nm for PAMAM G5.0-OH (). This can be attributed to the difference in architecture of linear HPMA copolymers that have a random coil conformation compared to hyperbranched PAMAM dendrimers which are more compact.
Figure 3 Size exclusion chromatograms of PAMAM dendrimers and HPMA copolymers using a Fast Protein Liquid Chromatography (FPLC) system with Superose 6™ 10/300 GL column (GE Healthcare, Piscataway, NJ) and an ultraviolet detector (GE Healthcare, Piscataway, (more ...)
Characterization of PAMAM dendrimers and HPMA copolymers
Depending on the chemical nature of the pendant side chains, linear polymers may possess a random-coil architecture in case of hydrophilic groups, an extended chain conformation for negatively charged moieties or a unimicellar folded structure in the case of hydrophobic side groups3
. The conformation of hyperbranched polymers such as PAMAM depends on generation with the smaller dendrimers having a flexible scaffolding and higher generations assuming a more compact, globular shape with a dense exterior and relatively hollow interior14
. The increment in size of polymers with increase in molecular weight is greater for HPMA homopolymer standards as compared to PAMAM-OH dendrimers (). However, below a molecular weight of about 29 kDa, the HPMA copolymers were smaller than the PAMAM-OH dendrimers of comparable molecular weight (). Above this molecular weight threshold of 29 kDa, the opposite trend was seen with the random coil HPMA homopolymer standards being larger in hydrodynamic size than the compact PAMAM-OH dendrimers ().
Correlation of molecular weight with hydrodynamic size of HPMA homopolymer standards and PAMAM dendrimers; Values are mean +/- S.D.; n=3.
The hydrodynamic sizes of amine-terminated dendrimers measured by dilute solution viscometry, light scattering, diffusion nuclear magnetic resonance and theoretically calculated by computer simulations that have been widely reported in literature are slightly higher than the hydrodynamic sizes measured for hydroxyl-terminated dendrimers of the same generation ()23-25
. This can be attributed to a more extended structure of the amine-terminated PAMAMs with charged surface groups as compared to neutral terminal groups in case of the hydroxyl terminated PAMAMs.
Upon comparing the sizes of the HPMA copolymers containing 20 mole % glycine-glycine ethanolamine and 2% tyrosine methyl ester with HPMA homopolymer standards containing 1 % pendant tyrosine groups, the HPMA copolymer of 26 kDa had a hydrodynamic radius of 1.4 nm, comparable to that of the HPMA homopolymer standard (22 kDa). The HPMA copolymer of 52 kDa, however, had a hydrodynamic radius of 3.3 nm, smaller than that of the HPMA homopolymer standard (51 kDa) with a hydrodynamic radius of 4.2 nm. This can be attributed to a higher tyrosine content in the HPMA copolymer of 52 kDa (0.22 mmol tyrosine/g polymer), as measured by amino acid analysis; than the theoretical tyrosine content in the HPMA homopolymer (0.07 mmol/g polymer) (). An increased number of tyrosine grafts on the HPMA copolymer backbone can lead to intra-molecular, hydrophobic interactions leading to a decrease in the hydrodynamic radius of the HPMA copolymer (52 kDa). Similarly, literature reported values of hydrodynamic radii of HPMA copolymers of similar molecular weights also vary depending on nature and percentage of side chains7
In vivo biodistribution
The smallest of the PAMAM dendrimers under study, G5.0-OH showed predominant and persistent accumulation in the kidney compared to all other organs (). G6.0-OH was taken up both by the kidney and the liver (). The extent and time of kidney accumulation was much less than G5.0-OH. However, it did not demonstrate extended circulation in the plasma and was taken up by the reticuloendothelial system (RES), thereby accumulating in the liver and spleen. G7.0-OH is known to have a rigid sphere like conformation14
. It had a hydrodynamic radius of 4.0 nm and showed the highest plasma circulation time (). This polymer was distributed over all organ systems due to retention in the plasma. Tumor accumulation profile was characteristic of macromolecules with slow accumulation that peaked at 6 hours and retained for 1 week. Small changes in hydrodynamic size of macromolecules in the nanometer range have been known to drastically affect pharmacokinetics26, 27
. MRI contrast agents based on PAMAM cores have shown a similar trend when increase in generation affected biodistribution, extravasation and mode of excretion26, 27
. PAMAM based gadolinium contrast agents have shown that constructs below 6.0 nm were predominantly excreted via the kidneys, while larger constructs were taken up by the liver instead26
. Those constructs in the size range of 5 to 8.0 nm were observed to extravasate into the tumor tissue from tumor vasculature. However, this data had limitations in quantitative interpretation owing to the detection technique (magnetic resonance). The size and conformation of the native PAMAM dendrimers of different generations may have also caused its interaction with plasma proteins, platelets and other components in the blood to differ. Detailed studies on interactions of these native PAMAM dendrimers with blood components could help better explain the effect of size and generation regarding its in vivo
Percentage of injected dose / g of tissue for G5.0-OH (Figure 5A, Top), G6.0-OH (Figure 5B, Middle) and G7.0-OH (Figure 5C, Bottom) in principal organs; Values are Mean +/- SEM; n=5; except n=4 for 5 min 2 hr G5.0-OH; 6 hour and 24 hr G7.0-OH.
HPMA copolymer of 26 kDa with the smallest hydrodynamic radius (1.4 nm) amongst the polymers tested, was excreted through the kidney () within 2 hours of administration and recovered in the urine (12% of injected dose, see Table 2 in supporting information
). It showed slight kidney accumulation (20 % injected dose/g) with slow renal clearance for up to 1 week (6 % injected dose/g). A neutral HPMA copolymer with 5 mol% GFLG-OH and 0.6 mol% tyrosine in the side chains has been reported to have a similar biodistribution profile in a tumor-bearing rat model8
. All major organ systems at 1 week were measured to have less than 1 % of injected dose/g including the kidneys8
. The difference in kidney accumulation between the reported HPMA-GFLG-OH copolymer and the HPMA copolymer under study can be attributed to the electronegative charge on the HPMA copolymer under study (zeta potential of -15.0mV, ). Reports in literature have demonstrated that anionized linear polymers accumulate in kidneys as a function of electronegative charge28, 29
. The HPMA copolymer of 52 kDa circulated in the plasma slightly longer than HPMA copolymer of 26 kDa and showed distribution in all organ systems (). It showed slight tumor accumulation that peaked at 6 hours but did not show prolonged retention at 24 hours. The tumor accumulation of the 52 kDa HPMA copolymer under study seemed less than that reported for HPMA homopolymer of similar molecular weight studied in a tumor-bearing rat model8
. The HPMA copolymer under study was seen to be eliminated through urinary excretion with 16 % of the injected dose in the urine 24 hours post injection (see Table 2 in supporting information
) and showed negligible liver accumulation. This can be explained due to the difference in hydrodynamic radius of the HPMA copolymer under study and the HPMA homopolymer standard as discussed in the polymer characterization section, thereby highlighting the importance of hydrodynamic size of the polymer in deciding the in vivo
Figure 6 Percentage of injected dose / g of tissue for HPMA copolymer, 26 kDa (Figure 6A, Top) and HPMA copolymer, 52 kDa (Figure 6B, Bottom) in principal organs; Values are Mean +/- SEM; n=5; except n=4 for 5 min HPMA copolymer (52 kDa) and 2 hr HPMA copolymer (more ...)
The filtration size cut-off for the kidney is known to range from a hydrodynamic diameter of 3.7 to 6.0 nm30
. PAMAM G5.0-OH and HPMA copolymer of 26 kDa (<5.0 nm in hydrodynamic diameter) can be readily filtered through the glomeruli. We observed kidney retention for G5.0-OH for 1 week up to 150 percent injected dose/gram of tissue (). However, HPMA copolymer of comparable molecular weight did not persist in the kidney for a week indicating that polymer conformation affected renal reabsorption and retention. Data in literature reports 80 percent of injected dose of PAMAM dendrimer, amine terminated, generation 4.0 (G4.0-NH2
)/gram of kidney tissue, which reduced to about 10 percent of injected dose/gram of kidney upon PEGylating the dendrimer31
. It has been reported that PAMAM G4.0-gadollinium complexes accumulate in the proximal straight tubules in the outer medulla stripe of the kidney32
. Limited mechanistic studies for renal retention of PAMAM dendrimers report the localization of these polymers in the lysosomes of proximal tubule cells32
. This uptake is only possible upon filtration of the dendrimers, providing access to reabsorbtion into the proximal tubules. The biodistribution of acetylated PAMAM G5.0 has been reported in literature and the construct has shown negligible kidney accumulation33
. Acetylation may increase the hydrodynamic size of the PAMAM beyond the glomerular filtration cutoff (> 5.0 nm) thereby denying access to proximal tubule cells for uptake. PAMAM G6.0-OH (Rh
= 3.0 nm, ) and G7.0-OH (Rh
= 4.0 nm, ) studied here are not readily filtered due to their hydrodynamic sizes being above the filtration threshold cutoff and hence do not show prolonged renal retention (). The renal uptake for PAMAM G7.0-OH is comparable to that of pegylated G4.0-NH2
dendrimers reported in the literature31
. This indicates that it is possible to reduce non-specific kidney uptake by increasing dendrimer generation to a hydrodynamic size beyond kidney filtration pore size cut-off. The smaller generation G5.0-OH that is observed to accumulate in the kidney may be used for kidney imaging to detect renal tubular damage32, 34
. The constructs suggested for this application have been dendrimer-based-magnetic resonance imaging (MRI) contrast agents that are amine terminated32, 34
. A hydroxyl-terminated dendrimer such as PAMAM G5.0-OH shows the same or higher extent of kidney accumulation and is likely to be less toxic in vivo
than the amine terminated dendrimer of same generation11
. Inspite of persistent kidney accumulation, PAMAM G5.0-OH did not show elevated kidney toxicity markers at 1 week (refer to supporting information, Figure 3A
). An elevated white blood cell count was observed (refer to supporting information, Figure 3B
). It has been observed that physicochemically modified HPMA copolymers accumulate in the kidney to a higher extent than their non-modified counterparts8
. The introduction of peptide moieties was found to increase kidney accumulation and a direct correlation was observed between the amount of carboxyl and hydrazide groups on the HPMA copolymer and the extent of kidney localization35
. HPMA copolymers functionalized with the targeting peptide RGDfK and DTPA (1,2-diamine-N,N-N′,N′,N″,N″-pentaacetic acid) chelating moiety in the side chains showed persistent kidney accumulation that correlated with the amount of peptide loading28
. It would be interesting to study the effect of surface modification of PAMAM dendrimers with peptides on kidney accumulation and establish a systematic correlation of the same in non-linear, hyperbranched polymers.
Figure 7 Percentage of injected dose / g of kidney tissue for PAMAM dendrimers and HPMA copolymers; Values are Mean +/- SEM, n=5; except n=4 for 5 min HPMA copolymer (52 kDa), 2 hr HPMA copolymer (26 kDa) and G5.0-OH; 6 hr and 24 hr G7.0-OH and n=3 for 1 wk HPMA (more ...)
PAMAM dendrimers (G5.0-OH and G6.0-OH) accumulated to a larger extent in the liver than HPMA copolymers of similar molecular weight (26 kDa and 52 kDa) (). PAMAM G6.0-OH, with a hydrodynamic radius of 3.0 nm () is on the threshold of kidney filtration size cutoff. Hence, its elimination is predominantly by the reticuloendothelial system (RES), with a high extent of accumulation observed in the liver (). PAMAM G7.0-OH with its rigid sphere-like globular structure and a hydrodynamic radius of 4.0 nm circulates longer in the plasma than PAMAM G6.0-OH (). Its liver accumulation is less than G6.0-OH at the time points under study. However, there is a possibility of an increase in RES organ uptake when the polymer may be eliminated from the tumor (beyond the range of the time points under study). It has been shown that amine terminated PAMAM Generation 4.0 and 5.0 interact with plasma proteins such as human serum albumin, bovine serum albumin and enzymes such as human erythrocyte acetyl cholinesterase and alter the protein conformation and enzyme activity36-40
. It has also been shown that in the case of other nanoparticulate constructs, smaller particles with higher surface curvature had a better retention of the native protein structure and function than larger nanoparticles41
. There is, therefore, a possibility that PAMAM G6.0-OH with a smaller hydrodynamic radius than G7.0-OH and a less dense surface exterior may interact differently with opsonizing proteins, causing it to be taken up by the liver. It would be necessary to investigate how the interaction of the PAMAM dendrimer with opsonizing proteins affects macrophage uptake and consequently in vivo
fate. Accumulation of PAMAM G6.0-OH up to 40% dose/gram of liver tissue did not affect liver function as indicative of plasma levels of alanine aminotransferase and aspartate aminotransferase (data not shown).
Figure 8 Percentage of injected dose / g of liver tissue for PAMAM dendrimers and HPMA copolymers, Values are Mean +/- SEM; n=5; except n=4 for 5 min HPMA copolymer (52 kDa), 2 hr HPMA copolymer (26 kDa) and G5.0-OH; 6 hr and 24 hr G7.0-OH and n=3 for 1 wk HPMA (more ...)
Tumor accumulation profiles are reflective of relative sizes as indirectly measured by size exclusion chromatography with the largest polymer PAMAM G7.0-OH (Rh
= 4.0 nm) showing the highest and most persistent tumor accumulation of about 4-6 percent injected dose/gram of tumor tissue up to a week (). As is characteristic of the enhanced permeability and retention effect, PAMAM G7.0-OH showed time-dependent accumulation in the tumor that peaked at 6 hours and persisted for up to 1 week with a tumor to blood ratio (T/B ratio) of about 12.7542, 43
. The extent of tumor accumulation of HPMA copolymer of 52 kDa was less than the HPMA homopolymer of comparable molecular weight reported in the literature8
. This can be attributed to intramolecular interactions amongst tyrosine-containing side chains (0.22 mmol/g of polymer) on the HPMA copolymer leading to a decrease in hydrodynamic radius (3.3 nm, ) as compared to 4.2 nm for the HPMA homopolymer standards. PAMAM G5.0-OH and HPMA copolymer of 26 kDa were cleared from circulation by kidney filtration and hence did not show tumor accumulation. The tumor accumulation data indicated a hydrodynamic radius cutoff of about 4.0 nm, below which prolonged tumor retention was not observed for the orthotopic ovarian carcinoma tumors under study. The HPMA copolymer of 52 kDa with a radius of 3.3 nm showed enhanced tumor accumulation up to 2 % injected dose/gram of tumor. However, it did not show enhanced retention. PAMAM G6.0-OH with a hydrodynamic radius of 3.0 nm did not passively target the tumor ().
Figure 9 Percentage of injected dose / g of tumor tissue for PAMAM dendrimers and HPMA copolymers; Values are Mean +/- SEM; n=5; except n=4 for 5 min HPMA copolymer (52 kDa), 2 hr HPMA copolymer (26 kDa) and G5.0-OH; 6 hr and 24 hr G7.0-OH and n=3 for 1 wk HPMA (more ...)
The plasma exposure of the polymers is consistent with the size exclusion profiles and hydrodynamic sizes of the macromolecules under study (). The largest carrier G7.0-OH (Rh = 4.0 nm) showed the highest plasma exposure, approximately 25 times that of the smaller polymers G5.0-OH and HPMA copolymer of 26 kDa (). The extended circulation time of the PAMAM G7.0-OH allowed enhanced tumor accumulation. Comparing the linear and branched polymers of similar molecular weights, HPMA copolymer of 52 kDa achieved a plasma exposure three times that of PAMAM G6.0-OH (58 kDa) (). This can be attributed to a slightly higher hydrodynamic radius of the HPMA copolymer (Rh = 3.3 nm) as compared to the compact dendrimer (Rh = 3.0 nm) that reduces kidney filtration. PAMAM G6.0-OH was also extensively taken up by the liver (), thus reducing its plasma circulation time.
Figure 10A Percentage of injected dose / g of plasma tissue for PAMAM dendrimers and HPMA copolymers, Values are Mean +/- SEM; n=5; except n=4 for 5 min HPMA copolymer (52 kDa), 2 hr HPMA copolymer (26 kDa) and G5.0-OH; 6 hr and 24 hr G7.0-OH and n=3 for 1 wk HPMA (more ...)
Area under the curve of plasma time course for PAMAM dendrimers and HPMA copolymers in percentage of injected radioactive dose.
The biodistribution results of the polymeric constructs under study have implications in the choice of carriers for drug delivery and imaging. Due to its selective and persistent kidney accumulation, PAMAM G5.0-OH should be used with caution to deliver drugs such as cisplatinum, methotrexate, stroptozotocin and mitomycin that cause kidney toxicity, but these polymers may be effectively used for kidney imaging to detect renal tubular damage and assess kidney function32, 34
. These polymers also have the potential to treat kidney diseases due to preferential renal targeting, which is an area of growing interest. PAMAM dendrimers accumulated in the liver to a higher extent than the HPMA copolymers under study and hence should be used with caution to deliver drugs like adriamycin, methotrexate, 6-mercaptopurine, carboplatin, L-asparaginase and pentostatin that can cause liver damage. High generation PAMAM G7.0-OH showed reduced non-specific uptake in the liver and kidney comparable to that of PEGylated dendrimers31
The effect of polymer architecture on drug loading, drug release, cellular delivery and pharmacological activity have been evaluated in vitro44, 45
. In this study, we have evaluated the influence of polymer architecture on in vivo
fate utilizing orthotopic A2780 ovarian tumor-bearing nude mouse models. Polymer architecture affected renal and hepatic uptake of the constructs under study. The branched PAMAM dendrimers accumulated to a larger extent in the liver than the linear HPMA copolymers. For polymers that could be easily filtered through the kidney, PAMAM dendrimers persisted for a longer period of time in the kidney tissue as compared to HPMA copolymers. This is indicative of a difference in the extravasation of polymers of varying architecture through fenestrations of healthy tissue46
. More work is needed to understand the effect of linear and hyperbranched polymer architecture on rates and extent of microvascular extravasation.