Bimodal Targeting Using Sulfonated, Mannosylated PEI for Combined Gene Delivery and Photodynamic Therapy

Article abstract of DOI:10.1111/php.12688

Photodynamic therapy (PDT) and gene delivery have both been used to target both cancer cells and tumor-associated macrophages (TAMs). Given the complex nature of tumor tissue, there could be merit in combining these strategies simultaneously. In this study, we developed a bimodal targeting approach to both cancer cells and macrophages, employing materials conducive to both gene delivery and PDT. Polymers libraries were created that consisted of cationic polyethyleneimine (PEI) conjugated to the photosensitizer pyropheophorbide-a, with sulfonation (to target selectin-expressing cells) and mannosylation (to target TAMs). Polyplexes, consisting of these polymers electrostatically bound to DNA, were analyzed for transfection efficacy and cytotoxicity toward epithelial cells and macrophages to assess dual-targeting. This study provides preliminary proof of principle for using modified PEI for targeted gene delivery and PDT.

Full text of DOI:10.1111/php.12688

Received Date : 05-Sep-2016
Revised Date : 26-Oct-2016
Accepted Date : 11-Nov-2016
Article type : Research Article
Bimodal Targeting Using Sulfonated, Mannosylated PEI for Combined Gene Delivery and
Photodynamic Therapy
Upendra Chitgupi1, Yi Li2, Mingfu Chen2, Shuai Shao1,2, Marie Beitelshees2, Myles Joshua Tan2,
Sriram Neelamegham2, Blaine A. Pfeifer2, Charles Jones*3, Jonathan F. Lovell*1,2
1Department of Biomedical Engineering, University at Buffalo, The State University of New York,
Buffalo, New York, USA.
2Department of Chemical and Biological Engineering, University at Buffalo, The State University of New
York, Buffalo, New York, USA.
3Abcombi Biosciences Inc., Buffalo, New York, USA.
*Corresponding authors’ emails: jflovell@buffalo.edu (Jonathan Lovell) and
charles.jones@abcombibio.com (Charles Jones)
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/php.12688
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ABBREVIATIONS
TAM = Tumor associated macrophage; MMP = Matrix Metalloprotenase; PEI = Polyethyleneimine; mx-
PEI = PEI with x% mannosylation; sx-PEI = PEI with x% sulfonation; Pyro = Pyropheophorbide-a; ADM
= Allyl-D-(+)-Mannose; PDT = Photodynamic Therapy; PSGL-1 = P-selectin glycoprotein-1; DMSO =
Dimethyl sulfoxide; DIPEA = N,N-Diisopropylethylamine; DCM = Dichloro methane; pDNA = plasmid
DNA; CHO = Chinese hamster ovary; CHO-L = L-selectin overexpressing CHO cells; CHO-P = P-
selectin overexpressing CHO cells; PBS = Phosphate buffer saline; FACS = fluorescence-activated cell
sorting; EDTA = ethylenediaminetetraacetic acid; MTT = (3-(4,5-Dimethylthiazol-2-yl)-2,5-
Diphenyltetrazolium Bromide); TGDP = Total gene delivery performance; eGFP = Enhanced green
fluorescent protein.
ABSTRACT
Photodynamic therapy (PDT) and gene delivery have both been used to target both cancer
cells and tumor-associated macrophages (TAMs). Given the complex nature of tumor tissue,
there could be merit in combining these strategies simultaneously. In this study, we developed a
bimodal targeting approach to both cancer cells and macrophages, employing materials
conducive to both gene delivery and PDT. Polymers libraries were created that consisted of
cationic polyethyleneimine (PEI) conjugated to the photosensitizer pyropheophorbide-α, with
sulfonation (to target selectin-expressing cells) and mannosylation (to target TAMs). Polyplexes,
consisting of these polymers electrostatically bound to DNA, were analyzed for transfection
efficacy and cytotoxicity towards epithelial cells and macrophages to assess dual-targeting. This
study provides preliminary proof of principle for using modified PEI for targeted gene delivery
and PDT.
INTRODUCTION
Macrophages engulf foreign bodies and serve important roles in the immune system and are
present in all human tissues. However, in the tumor microenvironment, tumor associated
macrophages (TAMs) are implicated in tumor metastasis, angiogenesis and tumor initiation,
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making them a target of interest.(1, 2) Macrophages can be classified as either M1 or M2
depending on their phenotype. M2 macrophages can provide a nutritional advantage to cancer
cells and promote angiogenesis, whereas M1 macrophages are generally associated with innate
immunity.(3) TAMs have served as a target for chemotherapeutic cargo delivery, imaging
agents, and vaccines.(4) It has been demonstrated that the controlled reprogramming of M2
macrophages to an M1 phenotype can induce toxicity to the surrounding cancer cells.(5) TAMs
secrete matrix metalloproteinases (MMP), which impact the tumor microenvironment.(4) For
example, it has been shown that MMP-7 expressed by TAMs cleaves Fas ligand, causing the
surrounding cells to become more resistant to cytotoxic drugs.(6) Thus, the ability to
downregulate MMP-7 expression via nucleic acid delivery could improve the efficacy of
chemotherapy. One mechanism that TAMs can be selectively targeted is via mannose receptors,
an approach which has been explored in the recent years.(7)
Photodynamic therapy (PDT) is a clinically approved therapeutic procedure that uses light of
specific wavelength and a photosensitizer to generate reactive oxygen species (ROSs), including
singlet oxygen which cause cell death via apoptosis or necrosis.(8, 9) However, mainstream
clinical applications of PDT for solid tumor treatment are limited by numerous factors including
limited light penetration in tissues, and also limited tumor tissue specificity and consequential
damage to adjacent healthy tissue. To improve specificity, extensive research efforts have
examined PDT using activatable and targeted photosensitizers.(10, 11) Not only do TAMs play a
role in PDT responses (12), but photosensitizers have been developed specifically to target
TAMs, using receptors including the scavenger receptor and the mannose receptor (CD206).(13-
15)
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With the long term goal of improving tissue specificity of PDT, we previously conjugated
the photosensitizer pyropheophorbide-α (pyro) to sulfonated polyethyleneimine (s-PEI), forming
pyro-s-PEI.(16) This allowed for the selective PDT in cells overexpressing selectins, which are
implicated in inflammation and cancer.(17, 18) As part of the inflammatory response under
chronic inflammatory conditions, specific leukocyte sub-populations infiltrate into the site of
inflammation through a cascade of events that is termed the ‘multistep leukocyte adhesion
cascade’. In the first step of this cascade leukocytes are captured and they roll on endothelial
surfaces that express P- and E-selectins, and also interact with other already recruited white
blood cells via L-selectin. P-selectin glycoprotein-1 (PSGL-1) is a well-known glycoprotein
ligand for L- and P-selectin, with other sialofucosylated glycoproteins also acting as ligands of
the selectins. Natural sulfated glycans are prominent entities that are well known to bind
selectins under physiological conditions. These molecules typically contain either 6-O-sulfate or
3-O-sulfate groups attached to either N-acetylglycosamine (GlcNAc) or galactose (Gal),
respectively. Prominent examples include GlyCAM-1(19) and sulfated L-selectin ligands that are
expressed on the high endothelial cells of lymph nodes.(20) While these entities do not, strictly
speaking, contain a sulfonate group (they are sulfates), both functional groups impart a local
negative charge that enables selectin-ligation by engaging the lectin domain of the protein. Thus,
they likely operate via the same mechanism. Such sulfated glycans bind L- and P-selectin with
similar affinity are the core-2 sialyl Lewis-X entity that is part of PSGL-1.(21) In general,
sulfonated macromolecules can bind L- and P-selectin.(16, 22, 23)
Previous studies have combined PDT with gene delivery.(24-26) PEI can be used gene
delivery and with an abundance of amine groups can readily be modified with targeting moieties.
Here, we explore the concept of using modified PEI conjugated to pyro for targeting TAMs and
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selectin-expressing cells. PEI allows packing of DNA for cell internalization and pyro confers
PDT capabilities. The polymer is modified with sulfate groups to target selectins and with
mannose groups to target the mannose receptor expressed on TAMs. The strategy of targeting
both TAMs and melanoma cells for PDT was recently reported using a viral method with cow-
pea mosaic virus and dendrons was based on the selective expression of vimintin on the
cells.(27)
MATERIALS AND METHODS
Materials. Branched PEI (10 kDa), 4-toluenesulfonyl chloride, D-(+)-mannose and other
chemicals were obtained from Sigma. O-benzotriazol-1-yl-
tetramethyluroniumhexaflurophosphate (HBTU) was obtained from VWR. Dialysis membranes
(3.5 kDa pore size) were obtained from Spectra/ Por.
Synthesis. Sulfonated-PEI was synthesized as described previously.(28) Briefly, 0.5 mmol
PEI was dissolved with vortexing in 50 mL of methanol. 8.5 mL (72 mmol) or 31 mL (266
mmol) of chlorosulfonic acid was added for the generation of 6% and 34% s-PEI respectively.
The reaction was maintained at 60 °C for 30 min. A thick yellow paste was obtained after
removing excess methanol and was dissolved in 5 mL water. The paste was vortexed until the
product formed a homogenous solution and was added to methanol to form a precipitate. This
was rinsed with methanol and the previous steps were thrice repeated in order to separate s-PEI.
s-PEI was placed in a vacuum desiccator for 24 h to remove solvents.
Allyl-α-d-mannopyranoside (ADM; mannose) was synthesized by mixing 3 g (16.6 mM) of
d-(+)-mannose with 8 mg (41.9 µM) toluenesulfonyl chloride in allyl alcohol (20 mL). The flask
was maintained at 90 °C for 24 h under reflux and the product was concentrated at 35 °C using
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vacuum distillation as previously described.(29) Mannosylated-PEI with varying mannose
percentages (6% and 34%) was synthesized as follows. PEI was dissolved in dimethyl sulfoxide
(DMSO) and allowed to react with varying amounts of ADM for 24 h at 90 °C to generate m-
PEI. Unreacted mannose was removed via dialysis. Pyro was then conjugated to s-PEI and m-
PEI as described below.
Pyro was synthesized as previously described.(30) 1 mL DMSO was added to PEI or s-PEI/
m-PEI and sonicated till the polymer completely dissolved. Pyro was added to DMSO containing
polymer, followed by HBTU and diisopropylethylamine (DIPEA) addition and stirring for 24 h.
Aqueous extraction was performed using 1:1 water and dichloromethane (DCM), and the
aqueous phase was retained. Aqueous extraction was performed thrice by retaining the aqueous
phase and adding equal volume of DCM to obtain pyro-labeled polymer. Small molecule
impurities were removed from the aqueous layer was via dialysis against water for 4 h with three
changes.
Gel shift assay. 500 ng of plasmid DNA (p-DNA) was mixed with indicated polymer in 25
mM NaOAc at pH 5.15 and the mixture was gently vortexed and incubated at room temperature
for 15 min. Samples were loaded on to a 0.8% agarose gel, which was then subjected to
electrophoresis and imaged with UV light and SYBR Safe DNA Gel Stain (Invitrogen). The
lowest concentration at which pDNA could be completely complexed with the polymer was
determined based on these results.
Polyplex characterization. Polyplexes were prepared by mixing polymer and pDNA in the
desired ratio (1:50, 1:100 and 1:200) in 25 mM NaOAc buffer at a pH of 5.15. A few modified
polymers, conjugated to mannose and sulfate, were not readily soluble so they were dissolved by
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sonication. Polyplexes were diluted in phosphate buffer saline (PBS) and the zeta potential and
size of the polyplexes was measured using Zetasizer nano-ZS90.
Cell viability assay and photodynamic therapy. Macrophages are known to overexpress
mannose receptors and have been used to study mannose binding.(29, 31, 32) Selectin
overexpressing cells, CHO-L and CHO-P, have been used to study sulfate binding.(16) CHO-L
and CHO-P cell lines were generated by transient transfection of Chinese Hamster Ovary (CHO)
cells with full length human L- and P-selectin. Individual clones were selected by serial dilution
and antibody selection.(21) These cells contain 200-500 selectins/µm2 as measured using
Quantum Simply Cellular cytometry calibration beads (Bangs laboratories) and flow cytometry.
Wild-type CHO cells, themselves, do not express either of the selectins. CHO and RAW 264.7
cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS
and 1% antibiotics. Cell densities for CHO and RAW 264.7 cells were 2x104 per well and 1x104
per well, respectively, and the cells were allowed to adhere for 24 h. In vitro experiments were
carried out in 96-well plates. For PDT experiments, media was replaced with polymer solution
(pyro concentration: 500 nM) diluted in PBS. Polymer treated wells were irradiated with laser to
determine targeting and PDT efficacy. A fluence of 10 J/cm2 (power density: 29.84 mW/cm2)
was achieved using a LED based 665 nm custom built laser box capable of irradiating the area of
the box with uniform power as previously described (16). 24 hours later, cell death induced by
PDT was measured using XTT assay. XTT (2, 3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-
Tetrazolium-5-Carboxanilide) and PMS (N-methyl dibenzopyrazine methyl sulfate) were diluted
in PBS to achieve a final concentration of 50 µg/mL and 30 µg/mL, respectively. Absorbance
values were read at 450 and 630 nm after incubating cells with 100µl of PBS containing XTT
and PMS for 3 h. Cell viability was calculated after subtracting the values at 630 nm from 450
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nm readings. The ratio of treated cells to untreated cells after subtracting XTT background was
used to determine the cell viability. All cell experiments were performed in triplicate and the
error bars represent standard deviation.
Cell transfection studies. Cell seeding densities for RAW264.7 and CHO cells in 96-well
plates were 30,000 and 20,000 per well, respectively. After 24 hours, media was removed and 30
µL polymer:pDNA polyplexes were added to each well containing 100 µL fresh media. Media
containing polyplex was replaced with fresh DMEM after 4 h and, 48 h later, gene delivery
efficiency was determined with the help of a flow cytometer. Luciferase expression level was
determined using Bright Glo Assay (Promega) and the total values were normalized to the total
protein in each well. Total protein in each well was determined using a luminometer, and was
calculated with the help of Micro BCA Protein Assay Kit (Pierce).
Transfection efficiency of the polyplexes was determined by calculating the number of
transfected cells using flow cytometry. 48 h post-transfection, media was removed and ice cold
PBS was used to rinse the wells in order to remove trace media before detaching the cells. Cells
were detached for fluorescence-activated cell sorting (FACS) analysis using either 0.05%
trypsin/ EDTA or cell scraper. Equal amount of DMEM containing serum was added in order to
deactivate trypsin. Cells were then resuspended in PBS after centrifuging at 250 g for 5 min.
Flow cytometer was used to determine the percentage of transfected cells.
MTT assay. Cells were seeded in a 96-well plate and allowed to adhere for 24 h. Cells were
transfected at the aforementioned concentrations and similar procedure for incubation were
followed. Media was replaced with 10% v/v 3-(4,5-Dimethylthiazol-2-yl)-2,5-
Diphenyltetrazolium Bromide (MTT) solution (5 mg/mL) diluted in PBS after 24 h. 100 µL of
MTT solution was added to each well and incubated for 3 h. MTT solution was replaced with
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DMSO to dissolve formazan crystals. After agitating gently for 1 h, the plate was read at 570 nm
using a microplate reader and 630 nm readings were used as background.
Measurements. NMR measurements were made with INOVA-500 in deuterated-DMSO at
500 MHz. A Zetasizer Nano ZS90 was used to determine the size and zeta-potential of the
polyplexes and a Synergy 4 Multi-mode Microplate Reader was used to measure the
luminescence in the well-plates. A Calibur flow cytometer was used for flow cytometry
measurements.
RESULTS AND DISCUSSION
Synthesis and characterization of polyplexes
Sulfate or mannose was conjugated to PEI via reaction with the amine groups of PEI as
shown in figure 1. The polymer itself is represented by a sphere in Figure 1 and a representative
segment is shown. Allyl-α-d-mannopyranoside was incubated with PEI overnight, allowing
amines to react with the monosaccharide. Sulfonated-PEI was produced by the addition of
chlorosulfonic acid to PEI dissolved in methanol, as previously described (16). Both s-PEI and
m-PEI were subjected to further purification steps in order to remove the impurities. Conjugation
of sulfate and mannose to PEI was confirmed by NMR (Figure S5). Pyro was conjugated to s-
PEI and m-PEI by dissolving polymer along with pyro in DMSO in the presence of HBTU and
DIPEA. Free carboxylic group on pyro reacts with primary amines on the surface of modified
polymers to generate pyro-labeled sulfated- or mannosylated-polymers. Post conjugation, pyro-
polymer conjugates were extracted using DCM-water extraction and then dialyzed overnight to
remove impurities. The number of pyro units was found to be between 2 and 4 per PEI polymer
based on pyro absorbance.
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To evaluate the formation of polymer-pDNA polyplexes, gel electrophoresis was performed
at different ratios (1:1 to 200:1) of polymer to pDNA (Figure S2-S4). Based on gel
electrophoresis, the minimum polymer concentration required for successful polyplex formation
was determined (table S1 and figures S2, S3, and S4) and the ratios 1:50, 1:100, and 1:200 were
used to study transfection efficiencies of the library of polymers. Polyplex size and zeta potential
were also measured (Figure S1). The average size of the polyplexes was found to be spread
between 100 and 350 nm. Pristine PEI polymers showed a mean diameter of 150 nm. However,
modified polymers showed variable size based on the conjugation ligand. With increasing
sulfonation, the size of the polyplex increased for s-PEI polymers (Fig S1A). At higher polymer
concentrations, these polyplexes tend to clump together due to increased electrostatic interaction,
which may explain the size increase. Although, pyro-labeled m6-PEI was an exception with
mean size ranging between 300 and 400 nm indicating they might have partially aggregated.
Zeta potential measurements showed that the positive charge on the polymer was only slightly
reduced with increasing sulfonation (Fig. S1B). However, the decrease in surface charge
rendered s-PEI polymers less toxic to both CHO (Figure 2) and RAW264.7 cells (Figure 5). This
might be explained by the fact that cationic groups on PEI are toxic to the cells, (33) and thus
modified PEI polymers with fewer primary amines show higher cell viability. Conjugation of
mannose to PEI did not have a marked influence on surface charge. Although no such trend was
observed with m-PEI polymers, unexpectedly, mannosyation seemed to increase the zeta
potential of the modified polymers. Moreover, conjugation of mannose to s-PEI polymers
showed higher positive charge overall compared to PEI or s-PEI polymers alone. The reason for
the increase in charge is not clear, purity of polymers or the interaction between DNA and
polymers might be the cause.
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Total gene delivery performance
Transfection magnitude or transfection efficiency alone cannot predict the performance of
the vector accurately. Therefore, to assess the gene delivery capabilities, the total gene delivery
performance (TGDP) has been used.(29, 34) Polymer modification in general had a positive
influence on the TGDP efficacy of the polyplexes. For example, at 1:50 polymer-pDNA ratio,
transfection efficiency of s34-PEI was 40% higher than PEI. Transfection magnitude, a measure
of total luminescence following luciferase transfection, presented similar trends, indicating
successful binding and uptake of polymers by selectin expressing cells. Although non-pyro-
labeled polymers showed similar luminescence values for CHO and CHO-L cells (expressing L-
selectin), CHO-P cells (expressing P-selectin) showed 4-5 fold higher transfection magnitudes
for sulfonated-polymers compared to unmodified PEI. Pyro-labeled polymers also exhibited
similar trends for eGFP transfection. Luciferase transfection increased with the sulfonation of
PEI. Transfection magnitude of sulfonated polyplexes increased by 4-5 folds for CHO-P and
CHO-L cells compared to CHO cells, indicating sulfonated polymers have better transfection
capabilities for selectin-expressing cells. Surprisingly, CHO cells exhibited an inverse relation
between polymer-pDNA ratio and transfection magnitude for pyro-labeled polymers. For
example, transfection magnitude for pyro-PEI at 50:1, 100:1 and 200:1 were 200, 100 and 75
RLU per µg of protein respectively. On the other hand, CHO-P and CHO-L cells displayed an
increase in transfection magnitude with increase in the concentration of the polymer.
In general, transfection efficiency and transfection magnitude showed pronounced effects in
CHO-P cells. In comparison, the transfection magnitude of CHO-L cells was lower than CHO-P
cells, suggesting that CHO-P cells might be binding sulfonated-polymers with higher specificity.
Although PEI has performed as an excellent transfection agent, it has been linked to higher
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toxicity and decreased cell proliferation.(33) Altering the surface amine groups of PEI with
anionic sulfate groups exhibited an increase in the cell viability suggesting modified PEI to be
less toxic compared to pristine PEI. Pyro conjugation to s-PEI enhanced the cell viability further,
which might be due to the decrease in amines on the surface of PEI/ s-PEI owing to the amide
bond formation between the carboxylic group of pyro and amine group of PEI.
Conjugation of mannose (6%) to PEI or s-PEI decreased the transfection efficiency in CHO
cells slightly but trends were similar to non-mannosylated polymers (figure 3). CHO cells
showed 50-60% transfection efficiency before mannose conjugation and 40-50% with m-PEI
polymer. CHO-L and CHO-P also displayed a decrease in transfection efficiency by 10-15% but
were generally higher than CHO cells showing sulfates to be still interacting with selectins after
mannose conjugation. However, luminescence assay results showed a drastic decrease in
transfection magnitude of CHO-P and CHO-L cells resulting in similar transfection magnitudes
for all three cell lines. m6-s34-PEI showed slightly higher transfection magnitude on CHO-L and
CHO-P cells indicating availability of excess sulfate moieties might overcome the hindrance due
to mannose modification. Mannosylation did not seem to affect pyro-labeled sulfonated-
polymers since they displayed similar transfection trends with eGFP and luciferase assay.
Mannosylation of PEI or s-PEI increased the cell viability at higher polymer-pDNA ratio but it
did not seem to have any effect on the cell viability at lower polyplex ratios.
Conjugation of higher percentage mannose (34%) to s-PEI polymers decreased the
transfection efficiency of the polymers on CHO cell lines (Figure 4). For example, if we compare
the performance of m34-s6-PEI versus s6-PEI on CHO and CHO-L cells, transfection efficiency
of CHO and CHO-L cells for m34-s6-PEI remained unchanged at approximately 40%, whereas s6-
PEI was nearly 25% higher for CHO-L cells at 1:50 polyplex ratio. Transfection magnitudes of
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s6-PEI and m34-s6-PEI showed similar trends in both CHO and CHO-L cells suggesting
transfection magnitudes to be unaffected by mannose modification. However, a comparison of
the same polymers on CHO and CHO-P cells suggested the transfection magnitude to be
drastically lowered by mannose conjugation. CHO-P cells showed a 4-folds higher transfection
magnitude with s6-PEI when compared with m34-s6-PEI, pointing towards lower binding and
therefore lower transfection. Although the modified polymers showed lower efficiency, pyro-
labeled polymers displayed identical trends with or without mannose modification. Moreover,
pyro-s6-PEI and pyro-m34-s6-PEI showed similar transfection magnitudes on CHO-P and CHO-L
cells. In general, pyro-conjugated polymers showed higher transfection efficiency with luciferase
assay compared to their non-pyro counterparts.
To verify if mannosylation would have any influence on transfection efficiency in
macrophages, we tested the gene delivery efficiency of mannosylated polymer libraries on
RAW264.7 cells. With increase in mannosylation, TGDP of the polymers improved, indicating
that binding and internalization was via mannose receptors on the surface of macrophages
(Figure 5). Transfection efficiency of mannosylated PEI (m-PEI) and s-PEI also showed a
marked improvement. Influence of mannosylation on transfection efficiency was verified by
comparing m6-PEI with m34-PEI. Increase in mannosylation from 6% to 34% resulted in 5%
improved transfection efficiency of m34-PEI. In comparison to PEI, at lower polymer-pDNA
ratio, m6-PEI and m34-PEI showed two and three fold increase in transfection efficiency
respectively. At 200:1 ratio, m34-PEI showed the highest EGFP transfection, but at the same time
it was also toxic with just 70% cell viability. Performance of pyro labeled m-PEI polymers
decreased slightly with increasing sulfonation at lower polymer-pDNA ratio. At 1:200 ratio
pyro-m34-s6-PEI performed the best with 40% transfection efficiency.
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The luminescence assay showed a slightly different trend for m-PEI polymers. Transfection
magnitude increased with increasing mannosylation percentage and the trend was clearly visible
at 1:200 polymer-pDNA ratio. However, the cell viability of m-PEI polymers decreased with
increasing polymer-pDNA ratio. At 1:50 ratio, cell viability of PEI polymers was nearly 100%,
upon increasing the concentration of the polymer in the polyplex, cell viability decreased by 20-
30% indicating unmodified PEI to be toxic at higher concentrations. Mannosylation alone did not
have much influence on cell viability however mx-sx-PEI polymers proved to be less toxic to
RAW264.7 cells even at higher concentrations. Pyro conjugation to mx-sx-PEI polymer reduced
their toxicity further. m34-PEI, which performed the best with respect to transfection efficiency,
had a TGDP value of 38. However, m34-PEI was toxic at higher polymer:pDNA ratios.
Considering all the metrics (cell viability, transfection magnitude, and efficiency) pyro-m34-s6-
PEI showed optimal performance on macrophages with a TGDP value of approximately 30.
In the absence of mannosylation, gene delivery efficiency of s-PEI polymers to macrophages
was low compared to their performance with CHO-P or CHO-L cells. Although s-PEI polymers
were less toxic to macrophages in comparison to PEI, eGFP or luminescence assays did not yield
high transfection efficiency on macrophages, even at higher polymer-pDNA ratios, leading to
poor TGDP (Figure 5). Previous studies have shown mannosylated polymers to undergo receptor
mediated endocytosis which accumulate in endosomes.(35, 36) Therefore, higher TGDP of m-
PEI polymers can be explained on the basis of improved targeting via mannose receptor. On the
other hand, s-PEI polymers displayed better PDT efficacy after short incubation but longer
incubation time did not translate to improved gene delivery. Clatherin mediated endocytosis
usually leads to a less efficient gene delivery owing to the degradation of polyplexes in
lysosomes.(37) Presence of lectins on the surface of cells might be leading to improved binding
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and uptake of sulfated polymers.(38) However, s-PEI polymers might be undergoing lysosomal
degradation after being internalized by macrophages leading to lower TGDP.
These results suggest that, in addition to dual-targeting capabilities, incorporating sulfate
and mannose on to a single polymer leads to higher transfection efficiency with improved cell
viability. However, while using dual targeting systems, studies involving a library of polymers
might be able to identify optimal system for gene delivery.
Photodynamic therapy
PDT with pyro-conjugated sulfonated and mannosylated polymers was assessed in vitro
with CHO, CHO-P, CHO-L, and RAW 264.7 cells. After a short 4 minute incubation of pyro-
labeled polymers with cells, they were irradiated with 665 nm light and cell viability was
assessed. Sulfonated-PEI polymers exhibited better targeting capabilities and hence more
efficient PDT with CHO-P and CHO-L cells compared to CHO cells (Figure 6). s-PEI polymers
showed marked reduction in cell viability for CHO-P, CHO-L, and RAW264.7 cells whereas
decrease in cell viability of cells incubated with pyro-PEI polymer was insignificant. Decreased
viability observed in CHO-P and CHO-L for pyro-m6-PEI can be explained based on the selectin
binding to sulfates on the surface of modified polymers. Macrophages might be binding to
lectins present on the surface of the leading to improved PDT efficacy.(38) In parallel, we
established the PDT efficacy of pyro-labeled mannosylated-PEI polymers on macrophages.
Since macrophages are known to express mannose receptors on their surface, we incubated
mannose and sulfonated polymer-pyro conjugates with RAW264.7 cells. Mannosylated-PEI has
been shown to effectively target mannose receptors.(39) Incubation of pyro-m6-PEI with
RAW264.7 cells followed by laser irradiation demonstrated higher cytotoxicity to macrophages,
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indicating successful targeting which corroborated with the previous studies. On the other hand,
CHO cells remained unaffected when treated with pyro-m6-PEI suggesting carbohydrate
targeting to be specific to RAW264.7 cells. These results collectively indicate that bimodal
targeting can be achieved in vitro.
Many studies have involved the use of either gene delivery or PDT as a standalone treatment
for therapy, synergistic use of PDT and gene delivery for therapy can enhance the efficacy of the
carriers. Some studies have shown the delivery of siRNA along with a photosensitizer enhances
the therapeutic efficacy for cancer therapy in vitro.(24) Other studies have demonstrated the use
of photosensitizer in light triggered release for gene delivery.(40) Thus, DNA packed polymers
conjugated to photosensitizers can serve multiple purposes.
CONCLUSION
A library of PEI polymers modified with varying amounts of mannose and sulfate moieties
was developed for targeted PDT and gene delivery. Conjugating a photosensitizer to sulfonated
and mannosylated polymers furnished them with PDT capabilities. Pyro-conjugated sulfate and
carbohydrate modified PEI polymers were able to induce cytotoxicity in selectin and mannose
receptor expressing cell lines thus exhibiting targeting and therapeutic capabilities. Modified
polymers enhanced the gene transfection efficiency and reduced cell toxicity compared to
unmodified polymer. Although higher percentages of sulfation and mannosylation resulted in
higher efficacy, trends were not clearly visible in a few polymers. Nevertheless, results from
these studies show that sulfonated and mannosylated polymers can serve as simultaneous targets
for epithelial cells and TAMs and also enable dual therapeutic modalities (PDT and gene
delivery).
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ACKNOWLEDGEMENTS
This work was supported by research funds from the National Institutes of Health
(DP50D017898 to J.F.L. and AI117309 to B.A.P.). M.C. is grateful to Mark Diamond Research
Fund for grant support.
SUPPORTING INFORMATION
Additional Supporting Information is available in the online version of this article or from
the author:
Figure S1. Polyplex characterization summary. Polyplexes were prepared at three
polymer:pDNA weight ratios and characterized using dynamic light scattering for A) particle
diameter (nm) and B) zeta potential (mV).
Figure S2. Gel shift assay of first generation polymers. Lane 1 is unbound polymer, whereas the
other lanes contain polymers. Specifically, L2 – 1:1, L3 – 5:1, L4 – 10:1, L5 – 25:1, L6 – 50:1,
L7 – 100:1, L8 – 200:1.
Figure S3. Gel shift assay of second generation polymers. Lane 1 is unbound polymer, whereas
the other lanes contain polymers. Specifically, L2 – 1:1, L3 – 5:1, L4 – 10:1, L5 – 25:1, L6 –
50:1, L7 – 100:1, L8 – 200:1.
Figure S4. Gel shift assay of third generation polymers. Lane 1 is unbound polymer, whereas the
other lanes contain polymers. Specifically, L2 – 1:1, L3 – 5:1, L4 – 10:1, L5 – 25:1, L6 – 50:1,
L7 – 100:1, L8 – 200:1.
Figure S5. 1H NMR spectra of all the polymers.
Table S1. Polymer:pDNA minimum binding ratio determined from gel electrophoresis.
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FIGURE CAPTIONS
Figure 1. Generation of sulfonated and mannosylated PEI. Step 1: Sulfonation of PEI. Step 2:
Pyro conjugation of s-PEI. Step 3: Synthesis of Allyl-α-d-mannopyranoside and mannosylation
of PEI/ s-PEI. Step 4: Pyro conjugation of mannosylated PEI/ s-PEI. The polymer is shown as a
sphere, the sulfonate groups in blue and mannose in red.
Figure 2. Effect of sulfonation (6% and 34%) of PEI on total gene delivery performance
(TGDP). A) CHO B) CHO-L (CHO cells expressing L-selectin) C) CHO-P (CHO cells
expressing P-selectin) transfected with polyplexes prepared at ratio of 50:1,100:1 and 200:1 of
polymer-pDNA. TGDP is the product of APC cell viability after transfection, transfection
efficiency (percent EGFP positive cells) and magnitude (luciferase). sx-PEI and pyro-sx-PEI
indicate sulfonated-PEI and pyro conjugated sulfonated-PEI respectively.
Figure 3. Effect of mannose conjugation (6%) of PEI on total gene delivery performance
(TGDP). A) CHO. B) CHO-L. C) CHO-P transfected with polyplexes prepared at ratio of
50:1,100:1 and 200:1 of polymer-pDNA. TGDP is the product of APC cell viability after
transfection, transfection efficiency (percent EGFP positive cells) and magnitude (luciferase).
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mx-PEI and pyro-mx-PEI indicate mannosylated-PEI and pyro conjugated mannosylated-PEI
respectively.
Figure 4. Effect of mannose modification (34%) of PEI on total gene delivery performance
(TGDP). A) CHO. B) CHO-L. C) CHO-P transfected with polyplexes prepared at ratio of
50:1,100:1 and 200:1 of polymer-pDNA. TGDP is the product of APC cell viability after
transfection, transfection efficiency (percent EGFP positive cells) and magnitude (luciferase).
Figure 5. Effect of sulfate and mannose modification of PEI on total gene delivery performance
(TGDP) of RAW264.7 cells. RAW264.7 cells were transfected with polyplexes prepared at three
polymer-pDNA weight ratios of A) sulfonation modified (pyro-) PEI, B) 6% mannosylation
modified sx-(pyro-) PEI, and C) 34% mannosylation modified sx-(pyro-)PEI. Subsequent TGDP
was calculated as the product of APC cell viability after transfection, transfection efficiency
(percent EGFP positive cells) and magnitude (luciferase).
Figure 6. Photodynamic Therapy. PDT cytotoxicity caused by pyro–labeled polymers in CHO
cells expressing P- or L-selectin and macrophages. Indicated polymers were incubated with cells
in a 96-well plate and then wells were irradiated with a fluence rate of 10 J/cm2. Cell viability
was assessed 24 h later using the XTT assay. Mean ± std. dev. for n = 4.
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.