Establishment of protein delivery systems targeting podocytes.
ABSTRACT Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease pathways can modulate glomerular function and influence severity and progression of glomerular disease.
Here, we describe studies that show efficient transport of proteins into the mammalian cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts caused an increase in DNA degradation. Moreover, purified proteins such as beta-galactosidase and the large GTPase dynamin could be profected into podocytes using two different profection reagents with the success rate of 95-100%. The delivered beta-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes. We are currently optimizing our protocol for in vivo profection.
Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as podocytes.
- [show abstract] [hide abstract]
ABSTRACT: Voltage-gated proton (H+) channels are found in many human and animal tissues and play an important role in cellular defense against acidic stress. However, a molecular identification of these unique ion conductances has so far not been achieved. A 191-amino acid protein is described that, upon heterologous expression, has properties indistinguishable from those of native H+ channels. This protein is generated through alternative splicing of messenger RNA derived from the gene NOH-1 (NADPH oxidase homolog 1, where NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate).Science 01/2000; 287(5450):138-42. · 31.03 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Cationic liposomal compounds are widely used to introduce DNA and siRNA into viable cells, but none of these compounds are also capable of introducing proteins. Here we describe the use of a cationic amphiphilic lipid SAINT-2:DOPE for the efficient delivery of proteins into cells (profection). Labeling studies demonstrated equal delivery efficiency for protein as for DNA and siRNA. Moreover, proteins complexed with SAINT-2:DOPE were successfully delivered, irrespective of the presence of serum, and the profection efficiency was not influenced by the size or the charge of the protein:cationic liposomal complex. Using β-galactosidase as a reporter protein, enzymatic activity was detected in up to 98% of the adherent cells, up to 83% of the suspension cells and up to 70% of the primary cells after profection. A delivered antibody was detected in the cytoplasm for up to 7 days after profection. Delivery of the methyltransferase M.SssI resulted in DNA methylation, leading to a decrease in E-cadherin expression. The lipid-mediated multipurpose transport system reported here can introduce proteins into the cell with an equal delivery efficiency as for nucleotides. Delivery is irrespective of the presence of serum, and the protein can exert its function both in the cytoplasm and in the nucleus. Furthermore, DNA methylation by M.SssI delivery as a novel tool for gene silencing has potential applications in basic research and therapy.Journal of Controlled Release 04/2007; 123:228-238. · 7.63 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Proteinuria is a major health-care problem that affects several hundred million people worldwide. Proteinuria is a cardinal sign and a prognostic marker of kidney disease, and also an independent risk factor for cardiovascular morbidity and mortality. Microalbuminuria is the earliest cue of renal complications of diabetes, obesity, and the metabolic syndrome. It can often progress to overt proteinuria that in 10-50% of patients is associated with the development of chronic kidney disease, ultimately requiring dialysis or transplantation. Therefore, reduction or prevention of proteinuria is highly desirable. Here we review recent novel insights into the pathogenesis and treatment of proteinuria, with a special emphasis on the emerging concept that proteinuria can result from enzymatic cleavage of essential regulators of podocyte actin dynamics by cytosolic cathepsin L (CatL), resulting in a motile podocyte phenotype. Finally, we describe signaling pathways controlling the podocyte actin cytoskeleton and motility and how these pathways can be manipulated for therapeutic benefit.Kidney International 11/2009; 77(7):571-80. · 7.92 Impact Factor
Establishment of Protein Delivery Systems Targeting
Wen Chih Chiang1,2, Tessa M. Geel3, Mehmet M. Altintas4, Sanja Sever1, Marcel H. J. Ruiters5, Jochen
1Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 2Department of Internal Medicine, National Taiwan
University Hospital, Taipei, Taiwan, 3Department of Pathology and Medical Biology, Groningen University Institute for Drug Exploration (GUIDE), University Medical
Center Groningen (UMCG), Groningen, The Netherlands, 4Division of Nephrology and Hypertension, Leonard Miller School of Medicine, University of Miami, Miami,
Florida, United States of America, 5Synvolux Therapeutics BV, Groningen, The Netherlands
Background: Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location
on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components
and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease
pathways can modulate glomerular function and influence severity and progression of glomerular disease.
Methodology/Principal Findings: Here, we describe studies that show efficient transport of proteins into the mammalian
cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures
that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake
of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are
physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts
caused an increase in DNA degradation. Moreover, purified proteins such as b-galactosidase and the large GTPase dynamin
could be profected into podocytes using two different profection reagents with the success rate of 95–100%. The delivered
b-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are
also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes.
We are currently optimizing our protocol for in vivo profection.
Conclusions: Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as
Citation: Chiang WC, Geel TM, Altintas MM, Sever S, Ruiters MHJ, et al. (2010) Establishment of Protein Delivery Systems Targeting Podocytes. PLoS ONE 5(7):
Editor: Vineet Gupta, University of Pittsburgh Medical Center, United States of America
Received January 5, 2010; Accepted July 2, 2010; Published July 29, 2010
Copyright: ? 2010 Chiang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: WC was sponsored by National Taiwan University Hospital and National Health Research Institute in Taiwan as well as by Divisional funds of the
Nephrology & Hypertension Division of the University of Miami. JR is supported by the US National Institutes of Health (NIH) grant DK073495. MMA was
supported by NIH training grant T32DK007540. MHJR is part of a commercial funder for this research. TMG also received materials from Synvolux Therapeutics to
perform parts of the described experiments. The funders contributed the study design, data collection and analysis.
Competing Interests: MHJR is employed by Synvolux Therapeutics and because of this company affiliation there is a competing interest on patents and
products of protein transfer tools described in this manuscript. The authors declare adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: email@example.com
Podocyte disease often results in proteinuria, a disease process
that can result from genetic mutations [1–5] or as the consequence
of signaling pathways that are in disarray due to the deleterious
action of cytosolic cathepsin L . In both settings, it is desirable
that podocytes can be reached by pharmacological interventions.
While it is possible to reach podocytes through gene transfer  or
small molecules , the delivery of functional peptides or proteins
inside podocytes have not been established yet. In order to
envision effective treatment for podocyte diseases, numerous drug
delivery approaches need to be established and potentially
combined. Moreover, protein delivery might be safer when
compared to gene or chemical therapy. Having such a cargo
system, it may allow the delivery of proteins or peptides into
podocytes that inhibits cytosolic cathepsin L activity or protect
cleavage targets from proteolysis. Podocytes are uniquely struc-
tured cells that are exposed to large quantities of both plasma and
urinary components. These characteristics predispose podocytes to
be amenable for protein delivery into intracellular space to
interfere with podocyte-specific disease pathways, modulate
glomerular function and alter the course of proteinuric renal
diseases. Our laboratory has developed a protein delivery protocol
that allows the uptake of exogenous protein that can exert its
function in multiple cells including podocytes.
DNA fragmentation after profection of NIH3T3 cells with
restriction enzyme MunI
To analyze if it was feasible to transport functional proteins into
cultured cells, we started out using mouse 3T3 fibroblasts that are
PLoS ONE | www.plosone.org1 July 2010 | Volume 5 | Issue 7 | e11837
known to be easily transfectable with plasmid DNA . We tested
the potential of transporting an apoptotic inducer such as the
restriction endonuclease MunI into the cells using SAINT-PhD as
a carrier. Forty-eight and 72 hours after profection, cells treated
with SAINT-PhD plus MunI showed decreased levels of genomic
DNA compared to control cells (untreated) or cell that received
SAINT-PhD only without MunI (Figure 1). These results indicate
that the transferred restriction enzyme MunI can exert its function
and cleave genomic DNA but only in the presence of the lipid
Profection of functional b-galactosidase in wild type
In our next sets of experiments, we utilized 2 different
profection reagents (SAINT-PhD and Chariot) as carriers to
analyze transport of b-galactosidase into differentiated podocytes.
Four hours after the application of the particles to the medium of
podocytes, the cells were incubated with X-gal substrate to
identify podocytes that have taken up enzymatically active b-
galactosidase. Both carriers allowed the transport of active b-
galactosidase into podocytes but with different efficiency. We
found larger than 90% blue cells using SAINT-PhD and about
80% of blue podocytes using Chariot (Figure 2). The difference
may lie in the different susceptibility of the carriers to serum.
Intracellular delivery ofproteins
significantly affected by the presence of serum as shown before
. However, in both cases, profection of active b-galactosidase
into podocytes was achievable with high yield and was detectable
even 4 days after profection.
by SAINT-PhDis not
Delivery of immunoglobulin with SAINT-PhD in
puromycin treated mouse podocyte
Since it is desirable to profect normal and diseased podocytes in
order to extend options for novel therapeutics we evaluated the
potential of antibody uptake in podocytes using SAINT-PhD and
IgG. Interestingly, we were unable to deliver unpacked IgG or
packed IgG into healthy podocytes but pretreatment of podocytes
for 24 hour with the nephrotoxic stimulant puromycin aminonu-
cleoside increased the uptake capacity for SAINT-PhD packed
IgG. Most of the transferred IgG shortly after uptake was located
in the membrane bound lysosomal compartment as seen by the co-
labeling of IgG with the lysosomal membrane marker LAMP2
Profection of dynamin into wild type podocyte
The large GTPase dynamin has recently been shown to be one
of the main targets of cytoplasmic cathepsin L leading to the foot
process effacement and proteinuria after being cleaved .
Stabilization of dynamin is thus one approach to rescue podocyte
function as shown by gene delivery of cleavage resistant dynamin
into mouse podocytes . Thus, we tested profection of full length
dynamin into cultured podocytes. Purified neuronal dynamin that
carries a His-tag (His-dyn1) was packed into SAINT-PhD carrier
and applied to the podocyte cell culture medium. His-tagged
dynamin was detected with an anti-His antibody inside podocytes
within 4 hours after profection (Figure 4). Of note, dynamin was
correctly sorted to plasma membrane and profection efficiency was
increasing with higher doses of purified dynamin packed in
Podocyte biology has made substantial progress over the
past few years ranging from ground braking genetic studies to
Figure 1. Restriction enzyme MunI delivery into mouse 3T3
fibroblasts. NIH3T3 mouse fibroblasts were seeded in a 48 well plate
(20,000 cells/400 ml per well) and treated with 0.5 mM MunI (48 h) or
0.05 mM MunI (72 h) alone or in complex with SAINT-PhD. Forty-eight
and 72 h after incubation cells were collected and DNA fragmentation
analysis was performed. BL: blank, untreated cells.
Figure 2. b-galactosidase was delivered into podocyte using SAINT-PhD and Chariot. Cells treated with only carrier agent (shown for
SAINT-PhD, A) or b-galactosidase without any carrier agent (B) did not show blue staining. The delivered enzyme exerted its function as it cleaved X-
gal substrate and turned the cells into blue color: Profection using SAINT-PhD (C) or Chariot (D) to carry b-galactosidase into podocytes.
Podocyte Protein Delivery
PLoS ONE | www.plosone.org2 July 2010 | Volume 5 | Issue 7 | e11837
detailed molecular analyses regarding the mechanisms of
proteinuria and podocyte injury . Novel therapeutic
approaches lie in the delivery of genes that encode for proteins
that are resistant during podocyte injury [6,11] or target
disease pathways with the use of small molecules [7,11]. An
additional approach would be to deliver entire and functional
proteins that can alter podocyte function under disease
conditions. In this study, we have investigated a novel
technique called profection. This technique uses lipid vesicles
as carriers. It is a simple technique that works well for cultured
podocytes. So far, the effects and efficiency vary for different
types of cargo proteins and we need to investigate further how
to optimize delivery and how to successfully use this approach
in vivo. Several agents have been developed to deliver protein
into cells, such as HIV-1 TAT , penetran 1  and VP22
. In addition, there is increasing use of nanogels carrying
proteins into tissues [15,16]. The above agents differ from
SAINT-PhD and Chariot in that they need to be covalently
bound to the macromolecules to be delivered . The
chemical reaction of covalent binding might denature the
protein to be delivered and inactivate the molecules. In
summary, we demonstrated that it is possible to deliver
functional proteins into cultured podocytes and other cells
using SAINT-PhD or Chariot. This approach bypasses the
transcription-translation process of gene therapy. However,
not all type of proteins can be transferred at equal amounts
and efficiency. With different proteins, the reagents have
different abilities to carry proteins into cells. Even the
condition of cells (diseased versus healthy) is a factor
influencing the uptake of the protein-carrier complex (e.g.,
IgG in this study). This study demonstrates the proof-of-
concept for SAINT-PhD or Chariot to deliver functional
proteins into podocytes and appears to be very promising for
future applications including in vivo studies. Cargo packed in
SAINT-PhD can result in sizes typically from 125–1700 nm
depending on cargo (protein size). The filtration barrier in the
kidney can classically restrict proteins or particles that are
larger than albumin because the pore sizes of the podocyte slit
membrane are roughly this size (4 nm by 14 nm). Based on this
size range, packed cargo is not routinely allowed to pass the
barrier. However, in glomerular kidney disease, there is a
breakdown of the size and charge-selective barrier and cargo
might pass the barrier relatively easily then. In addition, given
the strong endocytosis and pinocytosis capability of podocytes
, an uptake of cargo that is deposited in the glomerular
basement membrane is probably the main path of podocyte
entry. Our prediction would be that it is not required for cargo
to be filtered. Though in vitro results are encouraging, it will
require extensive research before effective in vivo therapies
using podocyte protein delivery systems might come into use
for treatment of podocyte-derived kidney diseases in humans.
Among the need to test profection in the living animal, it will
also become necessary to find out which factors influence
uptake of cargo to optimize intracellular delivery and
understand why not all cargo enter under the same conditions
(e.g., dynamin versus IgG).
Materials and Methods
Podocyte were cultured as previous described . Differentiated
wild type mouse podocytes (50,000 to 100,000 per well were
cultured on coverglasses and subjected to PBS or 50 mg/mL of
puromycin aminonucleoside (PAN) treatment for 24 hours.
Different amount of fluorescence labeled immunoglobulin IgG
(3–15 mg), His-tagged dynamin1 (1–3 mg), b-galactosidase (0.5–
3 mg, Active Motif, Carlsbad, CA, USA) were delivered into
podocyte using SAINT-PhD (Synvolux Therapeutics B.V., The
Netherlands) or Chariot (Active Motif) and packed as per provided
manufacturers’ protocols. As negative controls, dynamin protein
was also incubated without carrier at concentrations of 5–10 mg
protein/6 well. Mouse 3T3 fibroblasts were cultured in were
cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 50 mg/ml gentamycine sulfate, 2 mM L-
glutamine, 10% FBS.
For profection experiments, the cells were seeded in 48 well
plates and profected with 0.5 mM or 0.05 mM MunI the following
day. Forty-eight and 72 h after profection, the cells were harvested
and DNA fragmentation was carried out as described previously
. These protocols do not include covalent binding of cargo
protein to the carrier reagent. For Chariot profection, the ratio of
protein/reagent was 1–3/3.5 (mg/mL) in 0.5 mL of serum free
culture medium in a 12 well plate. Protein and reagent were mixed
and incubated at room temperature for 30 minutes. Then, the
mixture was added into the culture medium. Four or 48 hours
after profection, cells were fixed and labeled with suitable
antibody, examined by microscopy or subjected to FACS analysis
after labeling. Cells were examined with phase contrast or confocal
microscopy. b-galactosidase activity staining was performed using
the staining kit as protocol provided (Active Motif). DNA
fragmentation was analyzed with staining with propidium iodide,
followed by FACS examination.
Figure 3. Transfer of immunoglobulin into PAN-pretreated mouse podocytes. Cells treated with only carrier agent (shown for SAINT-PhD,
A) or IgG alone (B) did not have any uptake. In contrast, IgG packed into SAINT-PhD carriers allowed uptake of FITC-labeled IgG by podocytes. Cells
treated with SAINT-PhD and IgG (C) showed punctuated staining in cytoplasm (green) that co-localized in part with lysosomes which were labeled
with LAMP2 antibody (red) resulting a yellow overlap (D).
Podocyte Protein Delivery
PLoS ONE | www.plosone.org3 July 2010 | Volume 5 | Issue 7 | e11837
Wen Chih Chiang is a recipient of National Health Research Institute
Physician Scientist Award, Taiwan. The authors thank Prof. V. Siksnys,
Institute of Biotechnology, Vilnius, Lithuania for providing MunI.
Conceived and designed the experiments: WCC MMA SS MHJR JR.
Performed the experiments: WCC TG MMA SS. Analyzed the data:
WCC SS MHJR JR. Contributed reagents/materials/analysis tools: SS
MHJR JR. Wrote the paper: WCC MMA JR.
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