Dendrimer-inspired nanomaterials for the in vivo delivery of siRNA to lung vasculature

Article abstract of DOI:10.1021/nl5048972

Targeted RNA delivery to lung endothelial cells has the potential to treat conditions that involve inflammation, such as chronic asthma and obstructive pulmonary disease. To this end, chemically modified dendrimer nanomaterials were synthesized and optimized for targeted small interfering RNA (siRNA) delivery to lung vasculature. Using a combinatorial approach, the free amines on multigenerational poly(amido amine) and poly(propylenimine) dendrimers were substituted with alkyl chains of increasing length. The top performing materials from in vivo screens were found to primarily target Tie2-expressing lung endothelial cells. At high doses, the dendrimer-lipid derivatives did not cause chronic increases in proinflammatory cytokines, and animals did not suffer weight loss due to toxicity. We believe these materials have potential as agents for the pulmonary delivery of RNA therapeutics.

Full text of DOI:10.1021/nl5048972

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Communication
Dendrimer-inspired nanomaterials for the in
vivo delivery of siRNA to lung vasculature
Omar F. Khan, Edmond W. Zaia, Siddharth Jhunjhunwala, Wen Xue, Wenxin Cai, Dong
Soo Yun, Carmen Barnes, James E. Dahlman, Yizhou Dong, Jeisa M. Pelet, Matthew
J Webber, Jonathan Tsosie, Tyler Jacks, Robert Langer, and Daniel G. Anderson
Nano Lett., Just Accepted Manuscript • DOI: 10.1021/nl5048972 • Publication Date (Web): 19 Mar 2015
Downloaded from http://pubs.acs.org on March 23, 2015
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Dendrimerꢀinspired nanomaterials for the in vivo
delivery of siRNA to lung vasculature
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Omar F. Khan, Edmond W. Zaia, Siddharth Jhunjhunwala, Wen Xue, Wenxin Cai, Dong
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Soo Yun, Carmen Barnes, James E. Dahlman, Yizhou Dong, Jeisa M. Pelet, Matthew J.
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Webber, Jonathan K. Tsosie, Tyler E. Jacks,
Robert Langer,
Daniel G. Anderson.
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Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139, USA
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Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, USA
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Department of Anesthesiology, Children’s Hospital Boston, 300 Longwood Avenue, Boston,
MA 02115, USA
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Alnylam Pharmaceuticals, Cambridge, MA 02142, USA
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HarvardꢀMIT Division of Health Sciences and Technology, Institute for Medical Engineering
and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
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Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts
02139, USA
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Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge,
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Abstract
Targeted RNA delivery to lung endothelial cells has potential for treatment for conditions
that involve inflammation, such as chronic asthma, and obstructive pulmonary disease. To this
end, chemicallyꢀmodified dendrimer nanomaterials were synthesized and optimized for targeted
small interfering RNA (siRNA) delivery to lung vasculature. Using a combinatorial approach,
the free amines on multigenerational poly(amido amine) and poly(propylenimine) dendrimers
were substituted with alkyl chains of increasing length. The top performing materials from in
vivo screens were found to primarily target Tie2ꢀexpressing lung endothelial cells. At high
doses, the dendrimerꢀlipid derivatives did not cause chronic increases in proinflammatory
cytokines, and animals did not suffer weight loss due to toxicity.. We believe these materials
have potential as agents for the pulmonary delivery of RNA therapeutics.
Keywords
siRNA, target lung endothelial cells, in vivo, modified dendrimer nanoparticles, nanomaterial
Introduction
Pathologic gene expression in cells can be suppressed through RNA interference (RNAi).
RNAi is triggered when small interfering RNA (siRNA) for a specific gene is introduced into
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cells, which leads to the degradation of the corresponding mRNA gene transcripts.
To
enhance the usability of this naturally occurring phenomena for the treatment of lung diseases,
new materials must be developed to nanoencapsulate siRNA strands, deliver them to specific
lung cell subpopulations and avoid any nonꢀspecific or offꢀtarget effects. The preferential
targeting of lung endothelial cells is especially desirable for injuries and diseases that result in
lung vascular inflammation. Examples of such conditions include lung inflammation and
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reduced function after transplantation, barotrauma caused by the use of external ventilators, and
exacerbated pulmonary inflammation in asthmatics and chronic obstructive pulmonary disease
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COPD) patients. These conditions can lead to ischemic lung injury. The pulmonary
endothelium is an ideal target for therapeutic siRNA delivery because it not only controls
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vascular permeability, but can also modulate the innate immune response by releasing cytokines
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and recruiting circulating leukocytes.
The most advanced work in nanomaterialꢀbased siRNA delivery include hepatocyteꢀ
specific nanoparticles which have shown both selectivity and potency in nonꢀhuman primates
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and clinical trials.
While more challenging to achieve, there is an increasing collection of
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reports of siRNA delivery to tissues other than hepatocytes including tumors,
immune cells
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and endothelium. This work reports on the development of formulations based on dendrimeric
nanomaterials that have been optimized for lung endothelium delivery. The central hypothesis
was that nanoparticles created from symmetric, highlyꢀbranched ionizable nanomaterials are
capable of preferentially delivering siRNA payloads to the lung endothelium when administered
intravenously.
Branched polyethylenimines (PEI), which are ionizable polycations, have been used to
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successfully deliver siRNA payloads.
Randomly branched low molecular weight PEIs
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modified with alkyl chains have been developed for siRNA delivery and modifications using
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ethyl acrylate and succinic acid groups have also shown efficacy, though to a lesser extent.
However, while the PEI branching results in increased cationic charge density at low pH, it is not
molecularly defined; instead, it is a polydisperse polymer mixture with random branching. In
this way, dendrimers are advantageous because of their regulated, stepꢀwise growth and defined
branching patterns. While poly(amido amine) and poly(propylenimine) dendrimers have been
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used in both modified and unmodified forms to deliver siRNA,
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that result in preferential siRNA delivery to lung endothelial cells have not been reported.
Results
The chemicallyꢀmodified dendrimers were synthesized using Michael addition chemistry
by combining poly(amido amine) or poly(propylenimine) dendrimers of increasing generations
with alkyl epoxides of various carbon chain length (Scheme 1). The products were purified with
flash chromatography. The resulting product contained a mixture of different substitutions when
examined using thin layer chromatography (R between 0.4 and 0.8 for an 87.5∶11∶1.5
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CH Cl :MeOH:NH OH solvent system). The dendrimer cores are ionizable and cationic at
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physiological pH, which makes them ideal for forming complexes with negatively charged
siRNA. Initially, the chemicallyꢀmodified dendrimer nanoparticles were tested in vitro for Tie2
gene knockdown in both immortalized and primary endothelial cells. Tie2 was selected as a
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gene target because it is almost exclusively expressed by endothelial cells.
In these screens,
the only excipient used was 1,2ꢀdimyristoylꢀsnꢀglyceroꢀ3ꢀphosphoethanolamineꢀNꢀ
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methoxy(polyethylene glycol)ꢀ2000] (1,2ꢀdimyristoylꢀsnꢀglyceroꢀ3ꢀphosphoethanolamineꢀNꢀ
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mPEG2000). This was done to reduce the formulation complexity. Nanoparticles had a modified
dendrimer: Tie2 siRNA mass ratio of 5:1 and a 4:1 molar ratio of modified dendrimer to 1,2ꢀ
dimyristoylꢀsnꢀglyceroꢀ3ꢀphosphoethanolamineꢀNꢀmPEG2000. The chemicallyꢀmodified
dendrimers that displayed the best performance in primary endothelial cells (Figure 1a) were
then injected into the tail veins of healthy 8 week old female C57BL/6 mice. After 3 days, mice
were sacrificed and mRNA levels were quantified in the harvested tissues (Figure 1b). The top
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candidates from these screens were the generation 1 poly(amido amine) dendrimer with C15
lipid tails (PG1.C15), and generation 1 poly(propylenimine) dendrimers with C , C and C
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lipid tails (DG1.C14, DG1.C15 and DG1.C16, respectively).
Because the modified dendrimer materials contained a mix of dendrimer cores with
different levels of free amine alkyl chain substitution, the materials were further fractionated by
flash chromatography. This was done to determine which degree of substitution resulted in the
highest level of activity. The mixtures were initially divided into two fractions: R between 0.4
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and 0.6 for less substituted materials and R between 0.6 and 0.8 for more substituted materials
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87.5∶11∶1.5 CH Cl :MeOH:NH OH solvent system). The in vivo performance of the
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subfractions were compared to that of the mixture in terms of Tie2 knockdown. As seen in
Figure 2a, the mixture outperformed that of the fractions, implying that a broad range of
substitutions had a greater, synergistic effect.
To select the top 3 materials, nanoparticles that readily aggregated were removed from
contention. This was done to avoid injecting large nanoparticle aggregates into animals. The
propensity for aggregation was gauged by the magnitude of their zeta potential. Of the top 4
candidates, DG1.C16 had the least favorable (i.e. smallest) zeta potential, which predicted
aggregation (Table 1). As predicted, aggregation was observed when DG1.C16 particles were
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stored at 4°C. The aggregation was likely caused by the longer C lipid tail, which reduce
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overall solubility in aqueous environments. DG1.C14 and DG1.C15 both possess shorter lipid
chains, and so were more soluble. DG1.C16 was also redundant because the two remaining
poly(propylenimine)ꢀbased nanoparticles (DG1.C14 and DG1.C15) exhibited the same level of
protein knockdown as seen by Western blot (Figure 2b). Supporting Information (SI) Figure S1
shows the NMR spectra of the lead materials.
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To improve the efficiency of PG1.C15, DG1.C14 and DG1.C15 nanoparticles, the overall
formulation ratios and inclusion of additional excipients, such as cholesterol, were tested in vivo.
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many potent nanoparticle formulations.
It was discovered that the addition of cholesterol
improved in vivo Tie2 lung knockdown (Figure 3a). Moreover, the addition of cholesterol
appeared to alter the mean diameter and polydispersity index of the nanoparticles (Figure 3b and
SI Table S2).
To confirm knockdown was caused by the successful delivery of Tie2 siRNA and not due
to the modified dendrimer material itself, animals were treated with optimized modified
dendrimer formulations carrying nonꢀfunctional luciferase siRNA as a control. As expected,
these formulations resulted in no Tie2 gene knockdown (Figure 3c). Tie2 knockdown was also
examined in the endothelium of other organs but was most potent in the lung endothelium (SI
Figure S2). Figure 3d shows the structure of these optimized nanoparticles and their optimized
formulation ratios are shown in Table 2. Using Tie2 siRNA, a dose response was performed to
examine lowꢀdose efficiency (Figure 4a). Interestingly, when all three types of nanoparticles
were combined in a single dose, Tie2 knockdown in lung endothelial did not surpass the level of
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knockdown observed with individual nanoparticles. In terms of uptake mechanism, this may
indicate some type of molecular competition or blockage.
To qualitatively gauge the relative differences in lung delivery between the different
chemicallyꢀmodified dendrimers, nanoparticles containing Cy5.5ꢀlabelled siRNA were injected
into mice and their lung fluorescence imaged after 1 hour (Figure 4b). While DG1.C15
appeared to qualitatively show a greater amount of fluorescence, it was unclear if this was due to
increased endothelial cell delivery or delivery to other cells types within the lung. To address
this question, preferential nanoparticle association with lung endothelial cells was confirmed
using flow cytometry (Figure 5a, 5b and SI Figure S3). As an additional check, Tie2 siRNA
was replaced with Itgb1 siRNA (Figure 5c). Itgb1, or fibronectin receptor, beta subunit, is more
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ubiquitous than Tie2. It is an important protein in cellꢀmatrix adhesion, and it is expressed in
both the endothelial and epithelial cells of the lung. Thus, if nanoparticles were capable of
broader delivery, one would observe a greater amount of Itgb1 knockdown. Here, DG1.C14 and
DG1.C15 showed modest knockdown of Itgb1 (37 and 30%, respectively) and no knockdown
with PG1.C15. These results appeared to parallel the Cy5.5ꢀlabelled siRNA fluorescence data
where the lungs from DG1.C14 and DG1.C15 treatments showed more fluorescence than
PG1.C15 ones (Figure 4b). Hence, the greater amount of relative fluorescence in DG1.C14 and
DG1.C15 was attributed to an increased uptake in additional cell types.
Furthermore, as shown in Figure 5c, these nanoparticles were ineffective in terms of
siRNA delivery to epithelial lung tumors, which were generated by the targeted mutation of Kras
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and p53 genes in mice (KP mice). In this model, the lung tumor cells primarily expressed
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, and not the endothelial cells. Using this KP mouse lung tumor model, a previously
reported low molecular weight polyethyleneimineꢀbased polymer was capable of targeting both
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lung endothelial cells and epithelial lung tumor cells.
Moreover, previous studies have
suggested that tumors experience increased uptake due to the enhanced permeation and retention
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effect and their leaky vasculature. Thus, to test tumor uptake and gene silencing, KP mice
were treated with a high cumulative dose of 4 mg/kg Kras siRNA nanoparticles. Interestingly,
no Kras mRNA knockdown was found in the epithelial lung tumors. Thus, because of their
narrow targeting, these materials are primarily suitable for targeting lung endothelium and not
lung tumor cells.
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In addition to effective lung endothelial cell targeting, nanomaterials must not cause
chronic increases in proinflammatory cytokines. For example, in the case of human lung
transplantation, inflammatory cytokines such as TNFꢀα, IFNꢀγ, ILꢀ8, ILꢀ12 and ILꢀ18 are
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elevated, which can contribute to ischemic injury and diminish longꢀterm graft function. If
used to treat lungs post transplantation, the chemicallyꢀmodified dendrimers must not exacerbate
the inflammatory response by causing an increase in these cytokines. As a preliminary screen to
determine whether the chemicallyꢀmodified dendrimers cause a chronic increase in these and
other cytokines, plasma cytokine levels were measured 48 hours after high, medium and low
doses in mice (SI Figure S4). Fortuitously, no statistically significant increases in these and
other inflammationꢀrelated cytokines were observed. Additionally, no changes in mouse
bodyweights were observed when injected with high doses of the nanoparticles (SI Figure S5).
Discussion
The nanoencapsulation of siRNA and its successful delivery to lung endothelial cells in
vivo was achieved through the use of modified poly(amido amine) and poly(propylenimine)
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dendrimers substituted with hydrophobic lipid tails. The modification process resulted in a
mixture of substitutions. As seen in Figure 2a, nanoparticles created from a mixture of
substitutions outperformed those that were composed on smaller fractions, which implied that a
broad range of substitutions had a greater, synergistic effect. We believe the enhanced potency
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was caused by changes to the nanoparticles’ in vivo biomolecular corona, which in turn
affected trafficking into the endothelial cells. Nanoparticles formed from a mixture of modified
dendrimer molecules may adsorb different types or amounts of serum proteins. For example,
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nanoparticles coated with serum albumin have shown greater endothelial cell uptake in culture.
In a similar way, the optimized formulations that included cholesterol (Figure 3a) reduced the
overall amount of (1,2ꢀdimyristoylꢀsnꢀglyceroꢀ3ꢀphosphoethanolamineꢀNꢀmPEG2000), which is
used to mitigate protein adsorption to the particle. When coupled with the addition of
cholesterol, it is likely that more serum proteins were able to adsorb to the nanoparticle, which in
turn enhanced endothelial cell uptake.
To improve the potency of gene knockdown in lung endothelial cells, nanoparticle
formulations were optimized to include cholesterol, which markedly improved gene knockdown
efficiency (Figure 3a). However, some knockdown (albeit to a lesser degree) was still observed
in other organs at high doses (SI Figure S2), which means offꢀtarget effects in the endothelial
cells of other organs were not completely abolished. To compensate for this, lower doses should
be used.
Prior to optimization, DG1.C14 was the most potent nanoparticle. After optimization, the
diameters of PG1.C15 and DG1.C15 nanoparticles increased, while DG1.C14 did not (Figure 3b
and SI Table S2). Interestingly, PG1.C15 and DG1.C15 showed the greatest improvement in
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potency. This may indicate that each type of modified dendrimer nanoparticle has a critical
diameter for optimal endothelial cell delivery.
Furthermore, the chemical structure of these modified dendrimers are likely to play a role
in endothelial cell delivery and potency. The core of the PG1.C15 molecule has amide bonds,
which are not present in the DG1.C14 and DG1.C15 materials. These amide bonds may, at least
in part, be responsible for the narrower targeting window of PG1.C15 (Figure 5c).
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Additionally, the apparent nanoparticle pK values of these materials are different (Table
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these differences may also play a role.
Conclusions
In summary, siRNA formulated with chemicallyꢀmodified dendrimers have shown a high
avidity for Tie2ꢀpositive endothelial cells in the lung. The PG1.C15, DG1.C14 and DG1.C15
can be used to preferentially target endothelial cells within the lung. We believe these
formulations may have utility in the treatment of injuries and diseases that arise from
dysfunctional endothelium. Additionally, the use of molecularly defined, regularlyꢀbranched,
ionizable dendrimers as the core structure for these materials is advantageous with respect to
clinical translation. While demonstrated with siRNA here, we expect that these formulations of
these materials could have use for the delivery of other therapeutic nucleic acids, such as mRNA,
microRNA, and DNA.
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Methods
Alkyl epoxides: C , C , C and C epoxides were purchased from TCI or Sigma. C
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epoxide was not commercially available and was synthesized by the dropwise addition of 1ꢀ
pentadecene (TCI) to a 2x molar excess of 3ꢀchloroperbenzoic acid (Sigma) in dichloromethane
BDH) under constant stirring at room temperature. After reacting for 8 hours, the reaction
mixture was washed with equal volumes of super saturated aqueous sodium thiosulfate solution
Sigma) three times. After each wash, the organic layer was collected using a separation funnel.
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(
(
Similarly, the organic layer was then washed three times with 1 M NaOH (Sigma). Anhydrous
sodium sulfate was added to the organic phase and stirred overnight to remove any remaining
water. The organic layer was concentrated under vacuum to produce a slightly yellow,
transparent oily liquid. This liquid was vacuum distilled to produce the clear, colorless C15
epoxide liquid.
Modified dendrimer synthesis: Poly(amido amine) or poly(propylenimine) dendrimers of
increasing generations (Sigma Aldrich and SyMoꢀChem) were reacted with alkyl epoxides of
various carbon chain length. The stoichiometric amount of epoxide was equal to the total
number of amine reactive sites within the dendrimer (2 sites for primary amines and 1 site for
secondary amines). Reactants were combined in cleaned 20 mL amber glass vials. Vials were
filled with 200 proof ethanol as the solvent and reacted at 90°C for 48 hours in the dark under
constant stirring. The crude product was mounted on a Celite™ 545 (VWR) preꢀcolumn and
purified via flash chromatography using a CombiFlash Rf machine with a RediSep Gold
Resolution silica column (Teledyne Isco) with gradient elution from 100% CH Cl to 75∶22∶3
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CH Cl ꢁMeOHꢁNH OH (by volume) over 40 minutes. Thin layer chromatography (TLC) was
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used to test the eluted fractions for the presence of chemicallyꢀmodified dendrimers using an
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7.5∶11∶1.5 CH Cl ꢁMeOHꢁNH OH (by volume) solvent system. Chemicallyꢀmodified
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dendrimers with different levels of substitution appeared as a distinct band on the TLC plate.
The desired fractions were combined, dried under vacuum and stored under a dry, inert
atmosphere until used. All products contained a mixture of conformational isomers.
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Nanoparticle formulation: Tie2, Itgb1, CD45, luciferase and Alexa Fluor 647ꢀlabelled
GFP siRNA were supplied by Alnylam Pharmaceuticals and Kras siRNA was purchased from
Dharmacon. Because the mice used in these studies did not possess GFP and luciferase genes,
these siRNAs were used as a nonꢀfunctional controls. All siRNA sequences are shown in SI
Table S1. Nanoparticles were formulated using a microfluidic mixing device, as described
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elsewhere using the conditions found in Table 2. The 1,2ꢀdimyristoylꢀsnꢀglyceroꢀ3ꢀ
phosphoethanolamineꢀNꢀ[methoxy(polyethylene glycol)ꢀ2000] excipient was purchased from
Avanti Polar Lipids and cholesterol from Sigma. Nanoparticle size and zeta potential were
characterized with a Zetasizer NanoZS machine (Malvern). For accurate dilutions and dosage,
the concentration of siRNA was determined by a combination of Ribogreen assay (Invitrogen)
and NanoDrop measurement (Thermo Scientific). Apparent nanoparticle pK was determined as
a
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previously described. The specific doses for each experiment are listed in the main body of
this manuscript and are given as mg siRNA per kg mouse bodyweight.
In vivo animal experiments: 8ꢀ10 week old, female C57BL/6 mice were used in all
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experiments, with the exception of Kras experiments, where KP mice were used. All
procedures used in animal studies conducted at MIT were approved by the Institutional Animal
Care and Use Committee (IACUC) and were also consistent with all applicable local, state and
federal regulations.
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Flow cytometry: Mice were injected with a 2.5 mg siRNA/kg bodyweight dose of Alexa
Fluor 647ꢀtagged siRNA nanoparticles though the tail vein. One hour post injection, mice were
euthanized and their lungs were perfused with 15 mL of saline. Harvested lungs were minced
and digested for 1 hour at 37°C and 750 RPM in a PBS solution (Gibco) containing 0.92 M
HEPES (Gibco), 201.3 units/mL collagenase I (Sigma), 566.1 units/mL collagenase XI (Sigma)
and 50.3 units/mL DNase I (Sigma). Following digestion, cells and tissue debris were passed
through a 70 µm filter using a 1 mL syringe and the digestion buffer washed away by
centrifuging the single cell suspension at 400 RCF for 4 minutes at 4°C. Red blood cells in the
cell suspension were lysed using the ACK lysis buffer (Life Technologies, USA) and the
remaining cells suspended in flow cytometry buffer (PBS containing 0.5% BSA and 2mM
EDTA). For antibody staining, approximately 1 million cells were mixed with 100 µl flow
cytometry buffer containing antiꢀmouse CD45 (clone 30ꢀF11), antiꢀmouse CD31 (clone 390) and
antiꢀmouse EPCAM (clone G8.8) (Biolegend, San Diego, USA). Staining was performed at 4°C
for 20 min. Cells were suspended in flow cytometry buffer containing propidium iodide for
analysis. Flow cytometry was performed using a BD LSRꢀII and data analyzed using FlowJo
(Treestar Inc., USA). Endothelial cells were gated as CD31+ CD45ꢀ EPCAMꢀ, immune cells as
CD45+ CD31ꢀ EPCAMꢀ and epithelial cells as EPCAM+ CD31ꢀ CD45ꢀ.
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CD45 knockdown: 8ꢀ10 week old, female C57BL/6 mice (Charles River) were
intravenously injected via the tail vein with CD45 siRNAꢀcarrying nanoparticles at a dose of 1
mg siRNA/kg bodyweight. After 3 days, spleens were harvested, processed and analyzed as
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described previously.
Quantifying gene silencing: A QuantiGene 2.0 assay was used to quantify gene
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expression according to a previously published protocol. Kras silencing experiments were
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performed in KP mice at a total dose of 4 mg Kras siRNA/kg bodyweight. RTꢀPCR was used
to determine Kras silencing and data analyzed using the ꢂꢂC method.
T
Protein expression: Western blots were used to determine Tie2 protein expression levels
in lungs. Frozen pulverized lung tissue powder was dissolved in RIPA Lysis and Extraction
Buffer and Halt Protease and Phosphatase Inhibitor Cocktail (Pierce Biotechnology). Proteins
were extracted into the buffer at 4°C while mixing at 1400 RPM. Total protein concentration
was determined by the Pierce BCA protein assay kit (Thermo Scientific). Each lane of the preꢀ
cast MiniꢀPROTEAN TGX 4ꢀ15% polyacrylamide gradient gels (Bioꢀrad) was loaded with equal
amounts of total protein. After electrophoresis and transfer to nitrocellulose membrane (Bioꢀ
rad), equal total protein loading was confirmed by Ponceau S staining (Sigma). Blots were
blocked in Odyssey blocking solution and probed overnight with primary antibodies against Tie2
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(
R&D Systems) and βꢀactin (Sigma) followed by incubation in secondary LIꢀCOR antibodies
LIꢀCOR Biosciences). Blots were imaged using a LIꢀCOR Odyssey imaging system.
(
Cell culture: HMVECꢀL were cultured in EGMꢀ2 media (Lonza) and bEnd.3 in
Dulbecco’s Modified Eagle’s Medium supplemented with fetal bovine serum (ATCC). For in
vitro screening, cells were seeded and grown to confluence in tissue culture treated 96 well
plated. Cells were treated with nanoparticles at a 50 nM siRNA dose overnight before lysis and
mRNA quantification using the QuantiGene 2.0 Reagent System and QuantiGene 2.0 mRNA
probes (Affymetrix). Primary endothelial cells were used below passage 8.
Cytokine screen: Blood samples were collected at 2 days following tail vein injection of
modified dendrimer nanoparticles in C57BL/6 mice, and plasma was isolated for cytokine array
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screening using the BioꢀPlex Protein Array System (BioꢀRad) with a Luminex 200 instrument.
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TM
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The BioꢀPlex Pro Mouse Cytokine 23ꢀplex panel was combined with the BioꢀPlex Pro
Mouse Cytokine 9ꢀplex panel for an array totaling 32 cytokines.
Statistical analysis: Either ANOVA with the Tukey multiple comparison correction or
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the student tꢀtest was used to gauge statistically significant differences in mean values. P values
less than 0.05 were considered statistically significant. Statistical analysis was performed using
version 16 of the SPSS statistics software package.
ASSOCIATED CONTENT
Supporting Information
NMR spectra, organ screen data, immune cell data, blood inflammatory cytokine screen,
bodyweight data, siRNA sequences and full ANOVA table from library screen. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Correspondence should be addressed to Daniel G. Anderson; eꢀmail: dgander@mit.edu
Author Contributions
O.F.K., C.B. and J.E.D. conceived the modified dendrimer concept. O.F.K. designed
experiments. E.W.Z. assisted with animal experiments. E.W.Z and J.K.T. performed NMR.
D.S.Y. conducted highꢀresolution transmission electron microscopy. W.X. generated KP mice
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and W.C. performed the PCR reactions. M.J.W. performed inflammatory cytokine screen. S.J.
and J.M.P. assisted with flow cytometry studies. Y.D. provided assistance with material
purification. O.F.K. and D.G.A. primarily prepared the manuscript with input from all other
authors.
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Funding Sources
This work was funded by the National Institutes of Health Centers of Cancer and
Nanotechnology Excellence (U54 CA151884), the United States Army Medical Research and
Materiel Command’s Armed Forces Institute of Regenerative Medicine (W81XWHꢀ08ꢀ2ꢀ0034)
and Alnylam Pharmaceuticals. W.X. was supported by fellowships from the American
Association for Cancer Research and the Leukemia Lymphoma Society and is currently
supported by grant 1K99CA169512. M.J.W. received support from the Juvenile Diabetes
Research Fund. J.E.D. received support from the NDSEG, NSF GRFP, and the MIT Presidential
Fellowships.
Notes
R.L. is a shareholder and member of the Scientific Advisory Board of Alnylam. R.L and
D.G.A have sponsored research grants from Alnylam.
ACKNOWLEDGMENT
We expresses our gratitude towards J. Robert Dorkin, Hao Yin, Roman L. Bogorad and
Kevin J. Kauffman for useful advice and discussions. Tyler E. Jacks is a Howard Hughes
Investigator, the David H. Koch Professor of Biology, and a Daniel K. Ludwig Scholar.
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Animal experiments were performed at the Massachusetts Institute of Technology’s
Division of Comparative Medicine. Lung tissue processing and IVIS imaging was performed in
the Swanson Biotechnology Center in the Koch Institute for Integrative Cancer Research at the
Massachusetts Institute of Technology. We thank the Koch Institute’s Nanotechnology Materials
Lab for their cryogenic transmission electron microscopy service.
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Khan, O. F.; Zaia, E. W.; Yin, H.; Bogorad, R. L.; Pelet, J. M.; Webber, M. J.; Zhuang, I.; Dahlman, J.
E.; Langer, R.; Anderson, D. G., Ionizable amphiphilic dendrimer-based nanomaterials with alkyl-chain-
substituted amines for tunable siRNA delivery to the liver endothelium in vivo. Angew. Chem., Int. Ed.
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014, 53 (52), 14397-401.
40. DuPage, M.; Dooley, A. L.; Jacks, T., Conditional mouse lung cancer models using adenoviral or
lentiviral delivery of Cre recombinase. Nat. Protoc. 2009, 4 (7), 1064-1072.
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ranging in size from C to C were reacted with the free amines in poly(propylenimine) and
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Figure 1. Screening modified dendrimers for siRNA delivery and gene knockdown.
Poly(amido amine) and poly(propylenimine) products were denoted as PGx.Cy and DGx.Cy,
respectively, where x was the generation number and y the alkyl chain length. (a) Initial lowꢀ
generation in vitro modified dendrimer screen. Primary human microvascular endothelial cells
(HMVEC) from the lung and immortalized bEnd.3 endothelial cells from brain endothelioma
were treated overnight with modified dendrimer nanoparticles carrying Tie2 siRNA at a 50 nM
dose. Values are averages from duplicate experiments and are relative to PBS treated controls.
(b) In vivo lung screen with modified dendrimer nanoparticles formulated with Tie2 siRNA.
Mice were injected with a single 2.5 mg/kg Tie2 siRNA dose and gene expression was quantified
after 3 days. The top 3 performing materials were selected for further optimization. Gene
expression values are relative to PBSꢀtreated controls. N = 3 and error bars ± S.D.
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Figure 2. (a) The effect the degree of alkyl chain substitution has on nanoparticle performance.
Synergistic effects of modified dendrimer nanoparticles containing a range of alkyl chain
substitutions in the lung endothelium. The synthesis of the chemicallyꢀmodified dendrimers
resulted in a mixture of materials with different levels of substitution. The mixture (0.4 < R <
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days after a 2.5 mg/kg Tie2 siRNA nanoparticle dose. The full mixture containing the broadest
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range of substitution outperformed the subfractions. Gene expression values are normalized to
PBS treated controls. N = 3 and error bars + 1 S.D. Connecting lines indicate p < 0.009
(ANOVA with Tukey multiple comparison correction) for comparisons between the gene
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expression of the mixture and the subfractions. (b) Western blot showing a reduction in Tie2
protein expression in the lung 2 days after a 2.5 mg/kg Tie2 siRNA treatment with DG1ꢀclass
chemicallyꢀmodified dendrimers. DG1.C14, DG1.C15 and DG1.C16 all showed excellent
protein knockdown. N = 3.
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Figure 3. (a) Optimized modified dendrimer formulations. To tune delivery to lung endothelial
cells in vivo, cholesterol was added as a nanoparticle excipient. The additional excipient
improved Tie2 knockdown after a single Tie2 siRNA dose of 2.5 mg/kg. Gene expression values
are normalized to PBS treated controls. N = 3 and error bars ± S.D. * = p < 0.03 (tꢀtest) in
comparison to cholesterol use. (b) Nanoparticle size comparison with and without cholesterol.
The polydispersity in nanoparticle size decreased with the addition of cholesterol. For
nanoparticles containing C alkyl chains, the mean nanoparticle diameters also increased with
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the addition of cholesterol. (c) Negative controls for optimized modified dendrimer
formulations. No Tie2 knockdown was seen when animals were treated with chemicallyꢀ
modified dendrimers carrying nonꢀfunctional luciferase siRNA. N = 3 and error bars ± S.D. (d)
Cryogenic transmission electron microscope images of optimized PG1.C15 (left), DG1.C14
(middle) and DG1.C15 (right) nanoparticles. The dark regions that appear as folds or rings are
the internal lamellar structures of the nanoparticles. Scale bars = 50 nm.
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Figure 4. (a) Tie2 siRNA dose response using the top 3 nanoparticles, as well as a mixture of the
three. For these dose responses, the optimized, cholesterolꢀcontaining formulations were used
(refer to Table 2). Gene expression values are normalized to PBS treated controls. N = 3 and
error bars ± S.D. Connecting lines indicate p < 0.05 by ANOVA with Tukey multiple
comparison correction. (b) The relative, qualitative differences in lung fluorescence between
PG1.C15, DG1.C14 and DG1.C15 formulated with Cy5.5ꢀtagged siRNA. Modified dendrimer
nanoparticles were injected into the tail veins of mice and after 1 hour, lungs were harvested and
imaged. Total siRNA dose = 1 mg/kg and n = 3.
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Figure 5. (a) Preferential nanoparticle association and uptake in lung endothelial cells as shown
by flow cytometry. Chemicallyꢀmodified dendrimers were formulated with AlexaꢀFluor 647ꢀ
tagged siRNA and injected into the tail vein of mice. One hour postꢀinjection, lungs were
harvested and a single cell suspension was prepared for flow cytometry analysis. Endothelial
cells were identified as CD31+, CD45ꢀ and EPCAMꢀ, epithelial cells as EPCAM+, CD31ꢀ and
CD45, and immune cells as CD45+, CD31ꢀ and EPCAMꢀ. Grey curves are untreated controls
and white are nanoparticle treated. A large peak shift in fluorescent intensity was observed for
endothelial cells, which contrasted with the lack of shift in epithelial cells and a modest shift in
immune cells. (b) Quantification of nanoparticle association and uptake in terms of the Median
Fluorescent Intensity (MFI) for each treatment. Endothelial cells showed a statistically
significant increase in nanoparticle association and uptake, as compared to epithelial cells, for all
three nanomaterials. DG1ꢀclass materials also showed a significant increase in endothelial cell
uptake, as compared to immune cells. Connecting lines indicate p < 0.05 by ANOVA with
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