BACKGROUND
since its discovery in  <dig>  rna interference  has rapidly become a routine and powerful tool for use in basic research and has also gained momentum in development as a therapeutic  <cit> .
while rnai is an elegant, endogenous and conserved mechanism to selectively silence genes, inefficient delivery of exogenous short interfering rna  molecules to cells and tissues remains a barrier to its therapeutic development.
as a result, the design of effective sirna delivery systems is crucial for the clinical advancement of rnai.
specifically, delivery vectors must be designed to effectively complex with nucleic acid molecules and aid in overcoming intracellular barriers such as endosomal escape and cytoplasmic vector dissociation.
a variety of molecules including polymers, lipids, and peptides have been studied for their effectiveness as delivery vectors for dna and rna molecules  <cit> .
successful delivery vectors must exhibit a combination of functional attributes.
polymeric carrier molecules should be cationic to complex with nucleic acids, possess a high buffering capacity, exhibit low cytotoxicity, and also contain chemically reactive groups that can be modified for the addition of targeting moieties or other groups  <cit> .
highly branched, dendritic polymers including poly  have recently attracted interest as nucleic acid delivery vectors.
previous work has demonstrated that dendrimers can bind to dna and rna molecules and mediate modest cellular delivery of these nucleic acids  <cit> .
recently, some studies have evaluated the use of pamam dendrimers for successful delivery of sirna or antisense molecules.
generation  <dig> dendrimers were found to have poor cellular delivery of sirna to nih 3t <dig> mdr cells, though they mediated moderately effective delivery of antisense oligonucleotides  <cit> .
another study found that increasing the pamam dendrimer generation to seven to increase the number of primary amine groups significantly enhanced sirna delivery efficiency, possibly by enhanced amine-induced ph buffering <cit> .
however, cytotoxicity of highly cationic dendrimers is a marked problem that hinders their widespread use in drug and gene delivery  <cit> .
thus, it is desirable to exploit the potential of pamam dendrimers for nucleic acid delivery applications while reducing their cytotoxicity.
the cytotoxicity of dendrimers can be reduced by conjugating hydrophilic polymers to the periphery of the dendrimer  <cit> , by conversion of a fraction of the cationic amine groups to uncharged moieties  <cit> , or by modifying a neutrally charged dendrimer with a few cationic amino acid groups sufficient to facilitate nucleic acid complexation <cit> .
since a wide variety of modifications exist to alter the properties of dendrimers, the development of structure-activity relationships will accelerate determination of the optimal dendrimer properties for a particular application  <cit> .
in this study, the effect of primary amine acetylation of the cationic, dendrimeric polymer, pamam, on sirna delivery was analyzed.
previous studies have shown that neutralizing charges on the cationic polymer, polyethylenimine , by amine acetylation enhanced the transfection efficiency of plasmid dna  <cit> .
this marked improvement in transfection efficiency correlated with decreased polymer/dna interactions, thus promoting intracellular unpackaging of dna from the polymer  <cit> .
furthermore, amine acetylation of cationic polymers is attractive since it has been shown to reduce cytotoxicity in a variety of different cell lines  <cit> .
we studied dendrimer/sirna interactions as well as the ability of acetylated dendrimers to deliver sirna to cells and elicit a gene silencing effect.
in addition, we evaluated the tradeoff between reduced polymer/sirna interactions and reduced endosomal buffering capacity.
these design parameters are important in the rational modification of pamam dendrimers for sirna delivery.
RESULTS
partial acetylation of pamam dendrimers
the primary amines of generation  <dig>  pamam dendrimers were acetylated by reaction with prescribed amounts of acetic anhydride as depicted in figure  <dig>  1h nmr analysis of acetylated dendrimers was performed using a method similar to that described previously <cit> .
briefly, the fraction of primary amine acetylation was determined by comparing the intensity of the peak at  <dig>  ppm corresponding to -ch <dig> protons of the acetyl group to the sum of all -ch2-peaks.
the observed extents of amine acetylation were very close to the theoretical maxima indicated by reaction stoichiometry .
the acetylated dendrimers are denoted by their experimentally determined acetylation levels as ac <dig>  ac <dig>  ac <dig>  and ac <dig>
picogreen dye exclusion
the ability of acetylated dendrimers to complex with sirna as a function of polymer/sirna charge ratio was evaluated.
the amount of unbound sirna in solutions of dendrimer/sirna was determined by measuring the fluorescence of a commercially available dye, picogreen, that fluoresces upon binding to double-stranded dna or rna.
the fluorescence intensity decreased when increasing amounts of polymer were added to a fixed amount of sirna, indicating association of sirna with the polymer .
at n/p =  <dig>  differences in sirna complexation ability could be discerned, with higher fractions of sirna forming complexes with dendrimers possessing the greater primary amine levels.
however, by n/p =  <dig>  almost complete complexation of sirna was observed for unmodified dendrimer , ac <dig>  ac <dig>  and ac <dig>  in contrast, the binding curve for the ac <dig> polymer was shifted markedly to the right, and little sirna complexation was observed at n/p =  <dig>  since the ac <dig> dendrimer was not able to complex with sirna at n/p =  <dig>  it was excluded from further studies.
complex size and zeta potential
having demonstrated that acetylated dendrimers are able to complex with sirna, the characteristics of these complexes were evaluated further by dls and zeta potential measurements.
particle size analysis by dls showed the formation of ~ <dig> nm complexes between dendrimers and sirna, regardless of acetylation extent .
somewhat surprisingly, zeta potential measurements were approximately equal  for sirna complexes with g <dig>  ac <dig>  and ac <dig> dendrimers.
though we do expect the surface charge of these complexes to become less cationic upon primary amine acetylation, this trend may not be reflected in zeta potential measurements, as surface charge has been shown to be non-linearly correlated to zeta potential  <cit> .
however, while the zeta potential of dendrimer/sirna complexes at n/p =  <dig> for g <dig>  ac <dig>  and ac <dig> were approximately equal , the zeta potential did decrease somewhat for the ac60/sirna complex , indicating a modest change in the surface properties of this polyplex due to amine acetylation.
this trend is consistent with the dye exclusion results .
heparin induced polyplex dissociation
previous work has shown that more effective cellular delivery of nucleic acids is achieved when polymers are able to release or unpackage their cargo nucleic acid easily following cellular entry  <cit> .
to evaluate the effect of dendrimer amine acetylation on polyplex dissociation, dendrimer/sirna complexes were challenged by exposure to heparin sulfate, an anionic competitive binding agent.
polyplexes were formed in solution at n/p =  <dig> and exposed subsequently to either  <dig> μg/ml or  <dig> μg/ml of heparin sulfate.
after  <dig> minutes of incubation, picogreen was used to measure the amount of sirna released from dendrimer polyplexes.
as expected, more sirna was released from complexes with increasing heparin concentration .
upon addition of heparin, more sirna was released from  <dig> kda polyethylenimine , which was included as a well-studied reference, than from g <dig> dendrimer, indicating that 25k pei allows greater polyplex dissociation than g <dig> dendrimer.
consistent with the decrease in cationic charge density conferred by primary amine acetylation, the amount of sirna released increased with extent of primary amine acetylation.
at high fractions  of amine acetylation, the polyplex dissociation of acetylated dendrimers surpassed that of 25k pei.
cytotoxicity of acetylated dendrimers
primary amine acetylation of various cationic polymers has been shown to reduce their cytotoxicity  <cit> .
the cytotoxicity of acetylated dendrimers on a malignant glioma cell line, u <dig>  was evaluated by treatment with either dendrimer/sirna complexes  or with dendrimers in optimem medium for  <dig> hours .
subsequently, cells were analyzed for cell viability using the mts assay.
cells exposed to optimem medium alone were used as positive controls.
minimal cytotoxicity was observed from treatment with dendrimer/sirna complexes at typical cell transfection conditions .
however, it is also important to evaluate the cytotoxicity of the native polymers without the presence of sirna.
in this case, a dose-dependent cytotoxicity was observed by increasing the dendrimer concentrations to higher concentrations than were used for cell transfections .
additionally, a linear decrease in toxicity was observed upon reducing the number of primary amines by acetylation.
this trend is consistent with previous work that showed a linear decrease in cytotoxicity of a different cell type upon dendrimer amine acetylation  <cit> .
sirna induced silencing of gfp in u87-d1egfp cells
we tested the effectiveness of these polymers to deliver anti-d1egfp sirnas to u <dig> cells stably expressing the d1egfp transgene.
cells were treated with dendrimer/sirna complexes for  <dig> hours at several charge ratios within the range where all dendrimers  are able to complex the sirna.
after  <dig> and  <dig> hours, the d1egfp fluorescence of cells was analyzed using flow cytometry.
the fluorescence of polyplex treated cells was normalized to time-matched u87-d1egfp cells that received a mock treatment of serum-free medium .
sirnas delivered by unmodified g <dig> dendrimer produced significant gene silencing, manifest in reductions  of d1egfp fluorescence after  <dig> hours .
upon primary amine acetylation of dendrimers, a significant decrease in gfp silencing efficiency was observed, particularly for the dendrimer with the highest fractions of amine acetylation, ac <dig>  and ac <dig>  compared to unmodified dendrimer.
the ac <dig> dendrimer, with a modest fraction of amine modification, produced an insignificant change in gfp silencing ability as compared to the unmodified dendrimer .
for the g <dig> and ac <dig> materials, an improved gfp silencing ability was observed upon increasing the n/p ratio of each dendrimer from  <dig> to  <dig>  the extent and trends of gfp silencing using these polymers were maintained for  <dig> hours .
a low level of non-specific gfp silencing  was observed by all dendrimer/sirna complexes delivering a scrambled sirna sequence.
having studied the ability of acetylated dendrimers to deliver sirna to u87-d1egfp cells to elicit a gene silencing response, we used fluorescently labeled  sirna to determine intracellular levels of sirna delivered by the dendrimers to u <dig> cells that do not express d1egfp.
previous work has shown that efficient gene silencing correlates to high intracellular nucleic acid levels  <cit> .
a significant decrease in the intracellular fluorescence intensity of sirna was observed with increasing fractions of amine acetylation  .
this trend correlated closely to the decreased gfp silencing efficiency upon amine acetylation .
confocal imaging
confocal imaging was performed to compare the cellular distribution of nucleic acids delivered by acetylated dendrimers compared to unmodified dendrimers.
as endosomal escape is a well-known barrier to efficient nucleic acid delivery  <cit> , some transfections were performed in the presence of chloroquine diphosphate, a buffering agent, to identify if ph buffering is a significant barrier to efficient sirna delivery by acetylated dendrimers.
confocal imaging indicated that oligodeoxynucleotides  delivered by pamam dendrimers were sequestered into vesicles.
red odn fluorescence appeared as isolated, punctate specks when delivered by g <dig> or ac <dig> dendrimers , with somewhat greater cytoplasmic distribution observed for g <dig> delivery.
these distributions contrast with that of lipofectamine <dig>  a commercially available reagent, which delivered odn evenly throughout the cell .
further, the presence of chloroquine diphosphate during transfection mediated a homogeneous distribution of odn in a fraction of the cells when delivered by cationic dendrimers g <dig> , ac <dig> , or 25k pei .
titration of acetylated dendrimers
as the cellular distribution of odn delivered by dendrimers was improved by the presence of chloroquine in the transfection medium, it is likely that endosomal escape is a significant barrier to efficient sirna delivery for acetylated dendrimers.
hence, ph titrations were performed to compare the buffering capacity of acetylated dendrimers.
increasing the fraction of amine acetylation caused titration curves to shift, indicating a reduction in buffering capacity  upon addition of acid.
this trend is consistent with previous work performed with acetylation of pei  <cit> .
interestingly, the titration curve showed that unmodified g <dig> pamam has a much lower buffering capacity than  <dig> kda pei.
while pamam and pei have similar numbers of amines per unit mass, approximately half of the pamam amines are secondary amines in the form of amide bonds that are not titratable.
discussion
one motivation behind amine acetylation of pamam dendrimers was to promote polymer-sirna unpackaging in cells.
that is, self-assembled vectors for sirna delivery must associate strongly enough to remain intact during cellular binding and entry, yet they must dissociate at some point within cells to release their cargo.
previous studies have identified vector unpackaging to be an important barrier to polymeric gene and sirna delivery  <cit> .
specifically, correlations have been observed between the release of dna in competitive displacement assays in vitro and transfection efficiency in cells  <cit> .
furthermore, others have found that gene transfection was improved by increasing fractions of amine acetylation of pei, and this result was attributed to decreased polymer-dna interactions resulting in enhanced intracellular polyplex dissociation  <cit> .
previous work by our group has also reported a positive correlation between intracellular oligonucleotide levels  and enhanced complex dissociation among various molecular weights of pei  <cit> .
to reduce pamam/sirna interactions in this work, we employed partial amine acetylation of pamam dendrimers.
the heparin competition assay showed that unmodified generation  <dig> pamam dendrimer  exhibited less dissociation of sirna than did  <dig> kda pei.
given that 25k pei possesses a high charge density and exhibits strong association with oligonucleotides  <cit> , the even greater resistance of g <dig> pamam to heparin dissociation can be interpreted as indicating a poor tendency of the dendrimer to dissociate from sirna.
as hypothesized, greater dissociation was observed upon primary amine acetylation of pamam, which surpassed that of  <dig> kda pei at a high fraction  of primary amine acetylation.
however, polyplex size analysis demonstrated that stable particles of high positive zeta potential were formed between acetylated dendrimers and sirna, with a slight reduction in both at relatively high fraction  of primary amine acetylation.
this suggests that amine acetylation did not destabilize dendrimer/sirna complexes in isolation, but rather facilitated the ease of dissociation of sirna from the polymer complex in the face of an anionic competitor.
in addition to promoting polyplex dissociation by amine acetylation, we also expected to reduce cytotoxicity by partial charge neutralization.
cytotoxicity often correlates with a high charge density and is a marked problem with highly cationic pamam dendrimers.
previous work has shown a reduction in cytotoxicity upon amine acetylation of cationic polymers  <cit> .
indeed, we observed a linear decrease in cytotoxicity upon amine acetylation of pamam dendrimers.
thus, "tuning" of cationic polymer charge is a useful design principle in modulating both vector unpackaging and cytotoxicity.
another important barrier to the cellular delivery of nucleic acids is their sequestration in acidic endosomes.
cationic polymers with titratable amines are thought to mediate endosomal escape by buffering in the ph range of 5– <dig>  leading to osmotic pressure buildup and membrane permeability via the "proton sponge" effect  <cit> .
in the current study, we found that primary amine acetylation somewhat decreased the ph buffering capacity of dendrimers by removing primary amine groups.
this decrease in endosomal buffering capacity induced by amine acetylation is likely responsible for the decreased sirna delivery, as the presence of a buffering agent appeared to improve cellular distribution of oligonucleotides delivered by acetylated dendrimers.
it is important to note that acetylation of pei has been found to increase its ability deliver plasmid dna  <cit>  in contrast to the present study in which amine acetylation of pamam either did not affect  or decreased  the delivery of sirna.
since sirnas are much shorter and thus less polyvalent than plasmid dna, it is likely that sirna will more easily dissociate from a cationic polymer than would a large, anionic plasmid dna, making polyplex dissociation a potentially less critical step in the delivery process  <cit> .
further, it has been shown that the properties of polyplexes formed by dendrimers and rna depend significantly on the size of the rna molecule with more stable particles being formed with large rna molecules due to cooperative multivalent interactions <cit> .
these differences between polymers and their interactions with different types of nucleic acids highlight the importance of developing a more comprehensive and quantitative understanding of intracellular delivery mechanisms and their dependence on physicochemical attributes of the polymer and nucleic acid, so that data from various delivery systems and studies can be interpreted and used to rationally design nucleic acid carriers.
CONCLUSIONS
in summary, cationic dendrimers are promising architectures for use as nucleic acid delivery vectors.
their highly organized, cationic structure with functionalizable amine groups provides them with molecular properties that are favorable for efficient nucleic acid delivery.
neutralizing a fraction of primary amines by acetylation in this study did, as expected, promote polyplex unpackaging in vitro.
additionally, a reduction in cytotoxicity was noticed upon acetylation.
however, a reduction in ph buffering was also observed, possibly resulting in decreased sirna delivery to tumor cells.
the addition of a buffering agent chloroquine promoted favorable cellular distribution of nucleic acids delivered by dendrimers, indicating that endosomal escape is a substantial barrier to sirna delivery by these polymers.
others efforts to modify pamam dendrimers for gene or sirna delivery have also reported success in decreasing cytotoxicity but difficulties in achieving active nucleic acid delivery <cit> .
this observation demonstrates both the importance of endosomal buffering to sirna delivery as well as the advantages of charge reduction including reduced cytotoxicity and enhanced vector dissociation.
the next generation of dendrimers for sirna delivery will need to integrate charge reduction of dendrimers without compromising their endosomal buffering capacity.

 since its discovery in rna interference has rapidly become a routine and powerful tool for use in basic research and has also gained momentum in development as a therapeutic. while rnai is an elegant, endogenous and conserved mechanism to selectively silence genes, inefficient delivery of exogenous short interfering rna molecules to cells and tissues remains a barrier to its therapeutic development. as a result, the design of effective sirna delivery systems is crucial for the clinical advancement of rnai. specifically, delivery vectors must be designed to effectively complex with nucleic acid molecules and aid in overcoming intracellular barriers such as endosomal escape and cytoplasmic vector dissociation. a variety of molecules including polymers, lipids, and peptides have been studied for their effectiveness as delivery vectors for dna and rna molecules. successful delivery vectors must exhibit a combination of functional attributes. polymeric carrier molecules should be cationic to complex with nucleic acids, possess a high buffering capacity, exhibit low cytotoxicity, and also contain chemically reactive groups that can be modified for the addition of targeting moieties or other groups. highly branched, dendritic polymers including poly have recently attracted interest as nucleic acid delivery vectors. previous work has demonstrated that dendrimers can bind to dna and rna molecules and mediate modest cellular delivery of these nucleic acids. recently, some studies have evaluated the use of pamam dendrimers for successful delivery of sirna or antisense molecules. generation dendrimers were found to have poor cellular delivery of sirna to nih 3t mdr cells, though they mediated moderately effective delivery of antisense oligonucleotides. another study found that increasing the pamam dendrimer generation to seven to increase the number of primary amine groups significantly enhanced sirna delivery efficiency, possibly by enhanced amine-induced ph buffering. however, cytotoxicity of highly cationic dendrimers is a marked problem that hinders their widespread use in drug and gene delivery. thus, it is desirable to exploit the potential of pamam dendrimers for nucleic acid delivery applications while reducing their cytotoxicity. the cytotoxicity of dendrimers can be reduced by conjugating hydrophilic polymers to the periphery of the dendrimer, by conversion of a fraction of the cationic amine groups to uncharged moieties, or by modifying a neutrally charged dendrimer with a few cationic amino acid groups sufficient to facilitate nucleic acid complexation. since a wide variety of modifications exist to alter the properties of dendrimers, the development of structure-activity relationships will accelerate determination of the optimal dendrimer properties for a particular application. in this study, the effect of primary amine acetylation of the cationic, dendrimeric polymer, pamam, on sirna delivery was analyzed. previous studies have shown that neutralizing charges on the cationic polymer, polyethylenimine, by amine acetylation enhanced the transfection efficiency of plasmid dna. this marked improvement in transfection efficiency correlated with decreased polymer/dna interactions, thus promoting intracellular unpackaging of dna from the polymer. furthermore, amine acetylation of cationic polymers is attractive since it has been shown to reduce cytotoxicity in a variety of different cell lines. we studied dendrimer/sirna interactions as well as the ability of acetylated dendrimers to deliver sirna to cells and elicit a gene silencing effect. in addition, we evaluated the tradeoff between reduced polymer/sirna interactions and reduced endosomal buffering capacity. these design parameters are important in the rational modification of pamam dendrimers for sirna delivery.  partial acetylation of pamam dendrimers the primary amines of generation pamam dendrimers were acetylated by reaction with prescribed amounts of acetic anhydride as depicted in figure 1h nmr analysis of acetylated dendrimers was performed using a method similar to that described previously. briefly, the fraction of primary amine acetylation was determined by comparing the intensity of the peak at ppm corresponding to -ch protons of the acetyl group to the sum of all -ch2-peaks. the observed extents of amine acetylation were very close to the theoretical maxima indicated by reaction stoichiometry. the acetylated dendrimers are denoted by their experimentally determined acetylation levels as ac ac ac and ac picogreen dye exclusion the ability of acetylated dendrimers to complex with sirna as a function of polymer/sirna charge ratio was evaluated. the amount of unbound sirna in solutions of dendrimer/sirna was determined by measuring the fluorescence of a commercially available dye, picogreen, that fluoresces upon binding to double-stranded dna or rna. the fluorescence intensity decreased when increasing amounts of polymer were added to a fixed amount of sirna, indicating association of sirna with the polymer. at n/p = differences in sirna complexation ability could be discerned, with higher fractions of sirna forming complexes with dendrimers possessing the greater primary amine levels. however, by n/p = almost complete complexation of sirna was observed for unmodified dendrimer, ac ac and ac in contrast, the binding curve for the ac polymer was shifted markedly to the right, and little sirna complexation was observed at n/p = since the ac dendrimer was not able to complex with sirna at n/p = it was excluded from further studies. complex size and zeta potential having demonstrated that acetylated dendrimers are able to complex with sirna, the characteristics of these complexes were evaluated further by dls and zeta potential measurements. particle size analysis by dls showed the formation of ~ nm complexes between dendrimers and sirna, regardless of acetylation extent. somewhat surprisingly, zeta potential measurements were approximately equal for sirna complexes with g ac and ac dendrimers. though we do expect the surface charge of these complexes to become less cationic upon primary amine acetylation, this trend may not be reflected in zeta potential measurements, as surface charge has been shown to be non-linearly correlated to zeta potential. however, while the zeta potential of dendrimer/sirna complexes at n/p = for g ac and ac were approximately equal, the zeta potential did decrease somewhat for the ac60/sirna complex, indicating a modest change in the surface properties of this polyplex due to amine acetylation. this trend is consistent with the dye exclusion results. heparin induced polyplex dissociation previous work has shown that more effective cellular delivery of nucleic acids is achieved when polymers are able to release or unpackage their cargo nucleic acid easily following cellular entry. to evaluate the effect of dendrimer amine acetylation on polyplex dissociation, dendrimer/sirna complexes were challenged by exposure to heparin sulfate, an anionic competitive binding agent. polyplexes were formed in solution at n/p = and exposed subsequently to either μg/ml or μg/ml of heparin sulfate. after minutes of incubation, picogreen was used to measure the amount of sirna released from dendrimer polyplexes. as expected, more sirna was released from complexes with increasing heparin concentration. upon addition of heparin, more sirna was released from kda polyethylenimine, which was included as a well-studied reference, than from g dendrimer, indicating that 25k pei allows greater polyplex dissociation than g dendrimer. consistent with the decrease in cationic charge density conferred by primary amine acetylation, the amount of sirna released increased with extent of primary amine acetylation. at high fractions of amine acetylation, the polyplex dissociation of acetylated dendrimers surpassed that of 25k pei. cytotoxicity of acetylated dendrimers primary amine acetylation of various cationic polymers has been shown to reduce their cytotoxicity. the cytotoxicity of acetylated dendrimers on a malignant glioma cell line, u was evaluated by treatment with either dendrimer/sirna complexes or with dendrimers in optimem medium for hours. subsequently, cells were analyzed for cell viability using the mts assay. cells exposed to optimem medium alone were used as positive controls. minimal cytotoxicity was observed from treatment with dendrimer/sirna complexes at typical cell transfection conditions. however, it is also important to evaluate the cytotoxicity of the native polymers without the presence of sirna. in this case, a dose-dependent cytotoxicity was observed by increasing the dendrimer concentrations to higher concentrations than were used for cell transfections. additionally, a linear decrease in toxicity was observed upon reducing the number of primary amines by acetylation. this trend is consistent with previous work that showed a linear decrease in cytotoxicity of a different cell type upon dendrimer amine acetylation. sirna induced silencing of gfp in u87-d1egfp cells we tested the effectiveness of these polymers to deliver anti-d1egfp sirnas to u cells stably expressing the d1egfp transgene. cells were treated with dendrimer/sirna complexes for hours at several charge ratios within the range where all dendrimers are able to complex the sirna. after and hours, the d1egfp fluorescence of cells was analyzed using flow cytometry. the fluorescence of polyplex treated cells was normalized to time-matched u87-d1egfp cells that received a mock treatment of serum-free medium. sirnas delivered by unmodified g dendrimer produced significant gene silencing, manifest in reductions of d1egfp fluorescence after hours. upon primary amine acetylation of dendrimers, a significant decrease in gfp silencing efficiency was observed, particularly for the dendrimer with the highest fractions of amine acetylation, ac and ac compared to unmodified dendrimer. the ac dendrimer, with a modest fraction of amine modification, produced an insignificant change in gfp silencing ability as compared to the unmodified dendrimer. for the g and ac materials, an improved gfp silencing ability was observed upon increasing the n/p ratio of each dendrimer from to the extent and trends of gfp silencing using these polymers were maintained for hours. a low level of non-specific gfp silencing was observed by all dendrimer/sirna complexes delivering a scrambled sirna sequence. having studied the ability of acetylated dendrimers to deliver sirna to u87-d1egfp cells to elicit a gene silencing response, we used fluorescently labeled sirna to determine intracellular levels of sirna delivered by the dendrimers to u cells that do not express d1egfp. previous work has shown that efficient gene silencing correlates to high intracellular nucleic acid levels. a significant decrease in the intracellular fluorescence intensity of sirna was observed with increasing fractions of amine acetylation. this trend correlated closely to the decreased gfp silencing efficiency upon amine acetylation. confocal imaging confocal imaging was performed to compare the cellular distribution of nucleic acids delivered by acetylated dendrimers compared to unmodified dendrimers. as endosomal escape is a well-known barrier to efficient nucleic acid delivery, some transfections were performed in the presence of chloroquine diphosphate, a buffering agent, to identify if ph buffering is a significant barrier to efficient sirna delivery by acetylated dendrimers. confocal imaging indicated that oligodeoxynucleotides delivered by pamam dendrimers were sequestered into vesicles. red odn fluorescence appeared as isolated, punctate specks when delivered by g or ac dendrimers, with somewhat greater cytoplasmic distribution observed for g delivery. these distributions contrast with that of lipofectamine a commercially available reagent, which delivered odn evenly throughout the cell. further, the presence of chloroquine diphosphate during transfection mediated a homogeneous distribution of odn in a fraction of the cells when delivered by cationic dendrimers g, ac, or 25k pei. titration of acetylated dendrimers as the cellular distribution of odn delivered by dendrimers was improved by the presence of chloroquine in the transfection medium, it is likely that endosomal escape is a significant barrier to efficient sirna delivery for acetylated dendrimers. hence, ph titrations were performed to compare the buffering capacity of acetylated dendrimers. increasing the fraction of amine acetylation caused titration curves to shift, indicating a reduction in buffering capacity upon addition of acid. this trend is consistent with previous work performed with acetylation of pei. interestingly, the titration curve showed that unmodified g pamam has a much lower buffering capacity than kda pei. while pamam and pei have similar numbers of amines per unit mass, approximately half of the pamam amines are secondary amines in the form of amide bonds that are not titratable. discussion one motivation behind amine acetylation of pamam dendrimers was to promote polymer-sirna unpackaging in cells. that is, self-assembled vectors for sirna delivery must associate strongly enough to remain intact during cellular binding and entry, yet they must dissociate at some point within cells to release their cargo. previous studies have identified vector unpackaging to be an important barrier to polymeric gene and sirna delivery. specifically, correlations have been observed between the release of dna in competitive displacement assays in vitro and transfection efficiency in cells. furthermore, others have found that gene transfection was improved by increasing fractions of amine acetylation of pei, and this result was attributed to decreased polymer-dna interactions resulting in enhanced intracellular polyplex dissociation. previous work by our group has also reported a positive correlation between intracellular oligonucleotide levels and enhanced complex dissociation among various molecular weights of pei. to reduce pamam/sirna interactions in this work, we employed partial amine acetylation of pamam dendrimers. the heparin competition assay showed that unmodified generation pamam dendrimer exhibited less dissociation of sirna than did kda pei. given that 25k pei possesses a high charge density and exhibits strong association with oligonucleotides, the even greater resistance of g pamam to heparin dissociation can be interpreted as indicating a poor tendency of the dendrimer to dissociate from sirna. as hypothesized, greater dissociation was observed upon primary amine acetylation of pamam, which surpassed that of kda pei at a high fraction of primary amine acetylation. however, polyplex size analysis demonstrated that stable particles of high positive zeta potential were formed between acetylated dendrimers and sirna, with a slight reduction in both at relatively high fraction of primary amine acetylation. this suggests that amine acetylation did not destabilize dendrimer/sirna complexes in isolation, but rather facilitated the ease of dissociation of sirna from the polymer complex in the face of an anionic competitor. in addition to promoting polyplex dissociation by amine acetylation, we also expected to reduce cytotoxicity by partial charge neutralization. cytotoxicity often correlates with a high charge density and is a marked problem with highly cationic pamam dendrimers. previous work has shown a reduction in cytotoxicity upon amine acetylation of cationic polymers. indeed, we observed a linear decrease in cytotoxicity upon amine acetylation of pamam dendrimers. thus, "tuning" of cationic polymer charge is a useful design principle in modulating both vector unpackaging and cytotoxicity. another important barrier to the cellular delivery of nucleic acids is their sequestration in acidic endosomes. cationic polymers with titratable amines are thought to mediate endosomal escape by buffering in the ph range of 5– leading to osmotic pressure buildup and membrane permeability via the "proton sponge" effect. in the current study, we found that primary amine acetylation somewhat decreased the ph buffering capacity of dendrimers by removing primary amine groups. this decrease in endosomal buffering capacity induced by amine acetylation is likely responsible for the decreased sirna delivery, as the presence of a buffering agent appeared to improve cellular distribution of oligonucleotides delivered by acetylated dendrimers. it is important to note that acetylation of pei has been found to increase its ability deliver plasmid dna in contrast to the present study in which amine acetylation of pamam either did not affect or decreased the delivery of sirna. since sirnas are much shorter and thus less polyvalent than plasmid dna, it is likely that sirna will more easily dissociate from a cationic polymer than would a large, anionic plasmid dna, making polyplex dissociation a potentially less critical step in the delivery process. further, it has been shown that the properties of polyplexes formed by dendrimers and rna depend significantly on the size of the rna molecule with more stable particles being formed with large rna molecules due to cooperative multivalent interactions. these differences between polymers and their interactions with different types of nucleic acids highlight the importance of developing a more comprehensive and quantitative understanding of intracellular delivery mechanisms and their dependence on physicochemical attributes of the polymer and nucleic acid, so that data from various delivery systems and studies can be interpreted and used to rationally design nucleic acid carriers.  in summary, cationic dendrimers are promising architectures for use as nucleic acid delivery vectors. their highly organized, cationic structure with functionalizable amine groups provides them with molecular properties that are favorable for efficient nucleic acid delivery. neutralizing a fraction of primary amines by acetylation in this study did, as expected, promote polyplex unpackaging in vitro. additionally, a reduction in cytotoxicity was noticed upon acetylation. however, a reduction in ph buffering was also observed, possibly resulting in decreased sirna delivery to tumor cells. the addition of a buffering agent chloroquine promoted favorable cellular distribution of nucleic acids delivered by dendrimers, indicating that endosomal escape is a substantial barrier to sirna delivery by these polymers. others efforts to modify pamam dendrimers for gene or sirna delivery have also reported success in decreasing cytotoxicity but difficulties in achieving active nucleic acid delivery. this observation demonstrates both the importance of endosomal buffering to sirna delivery as well as the advantages of charge reduction including reduced cytotoxicity and enhanced vector dissociation. the next generation of dendrimers for sirna delivery will need to integrate charge reduction of dendrimers without compromising their endosomal buffering capacity. 
increasing amine acetylation resulted in reduced polymer cytotoxicity to u cells, as well as enhanced dissociation of dendrimer/sirna complexes. primary amine acetylation of pamam dendrimers reduced their cytotoxicity to u cells, and promoted the release of sirna from dendrimer/sirna complexes. in this work, the extent of primary amine acetylation of generation poly dendrimers was studied as a modification for the delivery of sirna to u malignant glioma cells. pamam dendrimers were reacted with acetic anhydride to obtain controlled extents of primary amine acetylation. acetylation of dendrimers reduced the cellular delivery of sirna which correlated with a reduction in the buffering capacity of dendrimers upon amine acetylation. a modest fraction of primary amines of pamam can be modified while maintaining the sirna delivery efficiency of unmodified pamam, but higher degrees of amine neutralization reduced the gene silencing efficiency of pamam/sirna delivery vectors. confocal microscopy confirmed that escape from endosomes is a major barrier to sirna delivery in this system. dendrimers with up to 60% of primary amines acetylated formed ~ nm complexes with sirna.
 the advancement of gene silencing via rna interference is limited by the lack of effective short interfering rna delivery vectors. rational design of polymeric carriers has been complicated by the fact that most chemical modifications affect multiple aspects of the delivery process. in this work, the extent of primary amine acetylation of generation poly dendrimers was studied as a modification for the delivery of sirna to u malignant glioma cells.  pamam dendrimers were reacted with acetic anhydride to obtain controlled extents of primary amine acetylation. acetylated dendrimers were complexed with sirna, and physical properties of the complexes were studied. dendrimers with up to 60% of primary amines acetylated formed ~ nm complexes with sirna. increasing amine acetylation resulted in reduced polymer cytotoxicity to u cells, as well as enhanced dissociation of dendrimer/sirna complexes. acetylation of dendrimers reduced the cellular delivery of sirna which correlated with a reduction in the buffering capacity of dendrimers upon amine acetylation. confocal microscopy confirmed that escape from endosomes is a major barrier to sirna delivery in this system.  primary amine acetylation of pamam dendrimers reduced their cytotoxicity to u cells, and promoted the release of sirna from dendrimer/sirna complexes. a modest fraction of primary amines of pamam can be modified while maintaining the sirna delivery efficiency of unmodified pamam, but higher degrees of amine neutralization reduced the gene silencing efficiency of pamam/sirna delivery vectors. 