21 research outputs found
Differential Polymer Structure Tunes Mechanism of Cellular Uptake and Transfection Routes of Poly(β-amino ester) Polyplexes in Human Breast Cancer Cells
Successful gene delivery with nonviral
particles has several barriers,
including cellular uptake, endosomal escape, and nuclear transport.
Understanding the mechanisms behind these steps is critical to enhancing
the effectiveness of gene delivery. Polyplexes formed with poly(β-amino
ester)s (PBAEs) have been shown to effectively transfer DNA to various
cell types, but the mechanism of their cellular uptake has not been
identified. This is the first study to evaluate the uptake mechanism
of PBAE polyplexes and the dependence of cellular uptake on the end
group and molecular weight of the polymer. We synthesized three different
analogues of PBAEs with the same base polymer poly(1,4-butanediol
diacrylate-<i>co</i>-4-amino-1-butanol) (B4S4) but with
small changes in the end group or molecular weight. We quantified
the uptake and transfection efficiencies of the pDNA polyplexes formulated
from these polymers in hard-to-transfect triple negative human breast
cancer cells (MDA-MB 231). All polymers formed positively charged
(10–17 mV) nanoparticles of ∼200 nm in size. Cellular
internalization of all three formulations was inhibited the most (60–90%
decrease in cellular uptake) by blocking caveolae-mediated endocytosis.
Greater inhibition was shown with polymers that had a 1-(3-aminopropyl)-4-methylpiperazine
end group (E7) than the others with a 2-(3-aminopropylamino)-ethanol
end group (E6) or higher molecular weight. However, caveolae-mediated
endocytosis was generally not as efficient as clathrin-mediated endocytosis
in leading to transfection. These findings indicate that PBAE polyplexes
can be used to transfect triple negative human breast cancer cells
and that small changes to the same base polymer can modulate their
cellular uptake and transfection routes
Uptake and Transfection with Polymeric Nanoparticles Are Dependent on Polymer End-Group Structure, but Largely Independent of Nanoparticle Physical and Chemical Properties
Development of nonviral particles for gene delivery requires
a
greater understanding of the properties that enable gene delivery
particles to overcome the numerous barriers to intracellular DNA delivery.
Linear poly(beta-amino) esters (PBAE) have shown substantial promise
for gene delivery, but the mechanism behind their effectiveness is
not well quantified with respect to these barriers. In this study,
we synthesized, characterized, and evaluated for gene delivery an
array of linear PBAEs that differed by small changes along the backbone,
side chain, and end group of the polymers. We examined particle size
and surface charge, polymer molecular weight, polymer degradation
rate, buffering capacity, cellular uptake, transfection, and cytotoxicity
of nanoparticles formulated with these polymers. Significantly, this
is the first study that has quantified how small differential structural
changes to polymers of this class modulate buffering capacity and
polymer degradation rate and relates these findings to gene delivery
efficacy. All polymers formed positively charged (zeta potential 21–29
mV) nanosized particles (∼150 nm). The polymers hydrolytically
degraded quickly in physiological conditions, with half-lives ranging
from 90 min to 6 h depending on polymer structure. The PBAE buffering
capacities in the relevant pH range (pH 5.1–7.4) varied from
34% to 95% protonatable amines, and on a per mass basis, PBAEs buffered
1.4–4.6 mmol of H<sup>+</sup>/g. When compared to 25 kDa branched
polyethyleneimine (PEI), PBAEs buffer significantly fewer protons/mass,
as PEI buffers 6.2 mmol of H<sup>+</sup>/g over the same range. However,
due to the relatively low cytotoxicity of PBAEs, higher polymer mass
can be used to form particles than with PEI and total buffering capacity
of PBAE-based particles significantly exceeds that of PEI. Uptake
into COS-7 cells ranged from 0% to 95% of cells and transfection ranged
from 0% to 93% of cells, depending on the base polymer structure and
the end modifications examined. Five polymers achieved higher uptake
and transfection efficacy with less toxicity than branched-PEI control.
Surprisingly, acrylate-terminated base polymers were dramatically
less efficacious than their end-capped versions, in terms of both
uptake (1–3% for acrylate, 75–94% for end-capped) and
transfection efficacy (0–1% vs 20–89%), even though
there are minimal differences between acrylate and end-capped polymers
in terms of DNA retardation in gel electrophoresis, particle size,
zeta potential, and cytotoxicity. These studies further elucidate
the role of polymer structure for gene delivery and highlight that
small molecule end-group modification of a linear polymer can be critical
for cellular uptake in a manner that is largely independent of polymer/DNA
binding, particle size, and particle surface charge
(3-Aminopropyl)-4-methylpiperazine End-capped Poly(1,4-butanediol diacrylate-co-4-amino-1-butanol)-based Multilayer Films for Gene Delivery
Biodegradable
polyelectrolyte surfaces for gene delivery were created through electrospinning
of biodegradable polycations combined with iterative solution-based
multilayer coating. Poly(β-amino ester) (PBAE) poly(1,4-butanediol
diacrylate-co-4-amino-1-butanol) end-capped with 1-(3-aminopropyl)-4-methylpiperazine
was utilized because of its ability to electrostatically interact
with anionic molecules like DNA, its biodegradability, and its low
cytotoxicity. A new DNA release system was developed for sustained
release of DNA over 24 h, accompanied by high exogenous gene expression
in primary human glioblastoma (GB) cells. Electrospinning a different
PBAE, poly(1,4-butanediol diacrylate-co-4,4′-trimethylenedipiperidine),
and its combination with polyelectrolyte 1-(3-aminopropyl)-4-methylpiperazine
end-capped poly(1,4-butanediol diacrylate-co-4-amino-1-butanol)-based
multilayers are promising for DNA release and intracellular delivery
from a surface
T Cell Targeting Biomimetic Polymeric Nanoparticles for mRNA Delivery and Stimulation
T cell immunotherapies have demonstrated robust clinical success in treating some cancers but are not without their challenges. Engineering T cells, such as chimeric antigen receptor (CAR) strategies, has been shown to be a powerful approach to direct the adaptive immune response in cancer. However, these approaches are expensive, time consuming, and inefficient given the need for T cell extraction from the patient, ex vivo engineering, then reinfusion. Genetically modifying T cells in situ would significantly simplify the process and reduce costs. Non-viral polymeric nanoparticles offer a potential vehicle for in situ gene engineering for their well-established efficacy and safety in multiple cell types. Here, we developed a modular, targeted polymeric/lipid NP that can deliver mRNA cargo and effectively stimulate T cells in situ. Using synthetic poly(beta amino ester) (PBAE), mRNA nanoparticles were synthesized by self-assembly in buffer. Particles were subsequently conjugated with either anti-CD3 or both anti-CD3 and anti-CD28, a costimulatory signal, to transfect primary murine T cells isolated from C57BL/6. Our results suggest that both in vitro and in vivo, mRNA-NPs with anti-CD3 conjugated to the surface significantly outperformed unconjugated NPs. In vitro, transfection efficacy of NPs + anti-CD3 reached ~17%, a 3-fold increase over the unconjugated particles. Likewise, particles conjugated with both anti-CD3 and anti-CD28 were able to induce ~5-fold T cell proliferation with minimal toxicity. For in vivo studies, transgenic Ai9 mice were injected systemically with mRNA-NPs encoding a Cre mRNA molecule for tdTomato expression analysis. NPs + anti-CD3 achieved significantly higher transfection in the spleen and lymph nodes than unconjugated particles, and preferentially transfected CD4+ T cells. Unconjugated particles had no significant preference for transfection in CD8+ or CD4+ T cells. Studies are currently underway to further investigate antibody conjugation and apply this T lymphocyte targeted gene delivery platform in situ for immunoengineering applications.
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Bioreducible Cationic Polymer-Based Nanoparticles for Efficient and Environmentally Triggered Cytoplasmic siRNA Delivery to Primary Human Brain Cancer Cells
siRNA nanomedicines can potentially treat many human diseases, but safe and effective delivery remains a challenge. DNA delivery polymers such as poly(β-amino ester)s (PBAEs) generally cannot effectively deliver siRNA and require chemical modification to enable siRNA encapsulation and delivery. An optimal siRNA delivery nanomaterial needs to be able to bind and self-assemble with siRNA molecules that are shorter and stiffer than plasmid DNA in order to form stable nanoparticles, and needs to promote efficient siRNA release upon entry to the cytoplasm. To address these concerns, we designed, synthesized, and characterized an array of bioreducible PBAEs that self-assemble with siRNA in aqueous conditions to form nanoparticles of approximately 100 nm and that exhibit environmentally triggered siRNA release upon entering the reducing environment of the cytosol. By tuning polymer properties, including bioreducibility and hydrophobicity, we were able to fabricate polymeric nanoparticles capable of efficient gene knockdown (91 ± 1%) in primary human glioblastoma cells without significant cytotoxicity (6 ± 12%). We were also able to achieve significantly higher knockdown using these polymers with a low dose of 5 nM siRNA (76 ± 14%) compared to commercially available reagent Lipofectamine 2000 with a 4-fold higher dose of 20 nM siRNA (40 ± 7%). These bioreducible PBAEs also enabled 63 ± 16% gene knockdown using an extremely low 1 nM siRNA dose and showed preferential transfection of glioblastoma cells <i>versus</i> noncancer neural progenitor cells, highlighting their potential as efficient and tumor-specific carriers for siRNA-based nanomedicine
Comparison of base polymer structure with reduction in metabolic activity.
<p>Each bar represents the average toxicity associated with the end-modified polymers that contained the base polymer shown (n = 11; error bar = SEM). Base diacrylate and side chain amino-alcohols are shown from least hydrophobic to most hydrophobic from left to right. (A) Reduction in metabolic activity of 30 w/w formulations averaged over 10 end-modified amines containing the base polymer shown. (B) Reduction in metabolic activity of 60 w/w formulations averaged over 10 end-modified amines containing the base polymer shown. For statistical analysis, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037543#pone-0037543-t001" target="_blank">Table 1</a>.</p
Number-averaged molecular weight versus time of B5-S5-E7 in PBS at 37°C with agitation.
<p>The half-life of the polymer in solution was 4.6 hr (R<sup>2</sup> = 0.984), and the polymer was almost completely degraded within 1 day.</p
Reduction in metabolic activity following PBAE nanoparticle administration.
<p>Formulations plotted at 0% reduction of metabolic activity here had equivalent or slightly higher metabolic activity than untreated controls. (A) Reduction in metabolic activity post transfection with polymer library formulated at 30 w/w ratio (n = 4). (B) Reduction in metabolic activity post transfection with polymer library formulated at 60 w/w ratio (n = 4).</p
Schematic showing polymerization scheme and monomers used.
<p>(A) Diacrylates (“B”) were added to primary-amine containing amino-alcohol side chains (“S”) to form the base polymers. (B) Base polymers were end-capped with amine monomers (“E”) to form the final, end-modified polymers. (C) The base diacrylate (“B”), amino-alcohol side chain (“S”), and end-modifying amines (“E”) used in the polymer library are listed here. (D) The full structure of B5-S5-E7 (1-(3-aminopropyl)-4-methylpiperazine-end-modified poly(1,5 pentanediol diacrylate-co-5-amino-1-pentanol) is shown here.</p
Results of 2-way ANOVA examining the effect of increased hydrophobicity of the side chain with respect to the base diacrylate it is paired with.
<p>NS is non-significant; P<0.05 is *; P<0.01 is **; P<0.001 is ***.</p