Targeted Delivery of siRNA to Activated T Cells via Transferrin-Polyethylenimine (Tf-PEI) as a Potential Therapy of Asthma

J Control Release. 2016 May 10;229:120-9.


Yuran Xie1, Na Hyung Kim1, Venkatareddy Nadithe1, Dana Schalk1,2, Archana Thakur1,2, Ayşe Kılıç3, Lawrence G. Lum1,2, David JP Bassett1, Olivia M Merkel1,2,4

1 Wayne State University, Detroit, MI, United States of America

2 Karmanos Cancer Institute, Detroit, MI, United States of America

3 Philipps-Universität Marburg, Marburg, Germany

Ludwig-Maximilians Universität München, Munich, Germany


Corresponding author:

Prof. Dr. Olivia Merkel

Department of Pharmacy

LMU München, Butenandtstr. 5-13 (Haus B), D 81377 München, Germany.

Tel. 089 2180 77025

FAX 089 2180 77020 



Asthma is a worldwide health problem. Activated T cells (ATCs) in the lung, particularly T helper 2 cells (Th2), are strongly associated with inducing airway inflammatory responses and chemoattraction of inflammatory cells in asthma. Small interfering RNA (siRNA) as a promising anti-sense molecule can specifically silence inflammation related genes in ATCs, however, lack of safe and efficient siRNA delivery systems limits the application of siRNA as a therapeutic molecule in asthma. Here, we designed a novel pulmonary delivery system of siRNA, transferrin- polyethylenimine (Tf-PEI), to selectively deliver siRNA to ATCs in the lung. Tf-PEI polyplexes demonstrated optimal physicochemical properties such as size, distribution, zeta-potential, and siRNA condensation efficiency. Moreover, in vitro studies showed significantly enhanced cellular uptake and gene knockdown mediated by Tf-PEI polyplexes in human primary ATCs. Biodistribution of polyplexes in a murine asthmatic model confirmed that Tf-PEI polyplexes can efficiently and selectively deliver siRNA to ATCs. In conclusion, the present work proves the feasibility to target ATCs in asthma via Tf receptor. This strategy could potentially be used to design an efficient siRNA delivery system for asthma therapy.

KEYWORDS:  Asthma; Polyethylenimine (PEI); Pulmonary delivery; Transferrin; siRNA delivery

PMID: 27001893



Asthma is a chronic pulmonary inflammatory disease and causes a worldwide health problem. Activated T cells (ATCs), particularly T helper 2 cells (Th2), orchestrate the development and amplification of inflammation in asthma. During activation and differentiation of Th2 cells, transcription factor GATA-3 is up-regulated and induce production of certain interleukins (e.g. IL-4, IL-5 and IL-13) which play key roles to promote inflammatory symptoms. Therefore, inhibit or silence the expression of GATA-3 could be an efficient treatment of asthma. The “drug” we propose to use is small interference RNA (siRNA) against GATA-3. siRNA was first described by Andrew Z. Fire and Craig C. Mello who won the Nobel Prize 2006 for this discover. It is an endogenous double stranded RNA and can efficiently and sequence-specifically silence target proteins. However, clinical application of siRNA has been hindered by the difficulty of its delivery. There are many challenges to deliver siRNA to targeted tissue/cells including instability of siRNA in blood stream, poor efficiency of crossing biological membranes and non-specific delivery to non-disease related tissue/cells. In this study, to overcome those challenges, we designed a pulmonary delivery system of siRNA, transferrin- polyethyleneimine (Tf-PEI), to target activated T cells in lung. We take advantage of the fact that PEI is an efficient vector to deliver siRNA and activated T cells overexpress the transferrin receptor. We hypothesized that Tf-PEI can selectively and efficiently deliver siRNA to activated T cells in vitro and in vivo.

In our study, we successfully conjugated Tf to PEI and characterized key physicochemical properties of Tf-PEI/ siRNA polyplexes including the size, surface charge and stability. The results showed that Tf-PEI/siRNA polyplexes demonstrated small size (<200 nm), slightly negatively surface charge and sufficient stability in the presence of lung surfactant and mucin. Small particles can be taken up by cells easier. Negative surface charge can reduce non-specific binding to the cellular membrane, which is also negatively charged, and consequently reduce potential side effects.

Next, we determined whether Tf-PEI can selectively deliver siRNA to T cells. Here, we have used two different cell models. We used primary human T cells from healthy human donors and activated them by ourselves. The other cell model is Jurkat cells which is an immortalized T cell line established from peripheral blood of an acute T cell leukemia patient. The high TfR expression status of both cell models was first confirmed. To study whether Tf-PEI has a better ability to deliver siRNA to TfR overexpressing cells compared with non-modified PEI, we treated both kinds of cells with Tf-PEI/siRNA polyplexes containing fluorescently labeled siRNA or with non-targeted PEI/siRNA polyplexes. The fluorescence intensity of polyplexes taken up into cells indicates the efficiency of delivery and can be quantified by flow cytometry. The results suggested that Tf-PEI can more efficiently deliver siRNA to both kinds of cells compared to PEI.

The cellular uptake of Tf-PEI/siRNA polyplexes is mediated by TfR, therefore, after internalized in to the cells, Tf-PEI/siRNA polyplexes will first be located in the endosome, a membrane vesicle. siRNA can silence genes once it is in cytoplasm, thus, one of the biggest challenges of siRNA delivery is the escape from the endosome. To investigate whether siRNA could be released from endosomes and achieve gene silencing, both kinds of cells were treated with Tf-PEI/siRNA polyplexes containing siRNA against GAPDH, a housekeeping gene expressed universally in cells, or PEI/siRNA polyplexes. The results indicated that Tf-PEI can achieve better gene silencing in both kinds of cells than PEI.

All in vitro results were very promising. Therefore, the next question we asked was whether these Tf-PEI/siRNA polyplexes really target activated T cells in vivo when administrated through lung. Therefore, we determined the biodistribution of Tf-PEI/siRNA polyplexes containing fluorescently labeled siRNA, which are easily detected, in a mouse asthma model. To establish a mouse asthma model, mice were sensitized by intraperitoneal injection of ovalbumin (OVA), a protein from chicken egg, followed by challenge of inhaled OVA aerosol as shown in Figure 1. The exposure of OVA will cause an inflammatory response in the lung which can mimic asthma symptoms. To administer Tf-PEI/siRNA polyplexes or PEI/siRNA polyplexes, animals were anesthetized and polyplex solutions were intratracheally instilled to the lung as shown in Figure 2.

 Figure 1 Inhalation of OVA aerosol setup for animals. The bottle under the platform is filled with OVA/saline solution. Air flow from the yellow tubing aerosolizes the OVA solution and delivers an OVA mist to the big chamber on top. Animals in tubes are forced to inhale the aerosol from the big chamber.


To evaluate the asthma response and the biocompatibility of Tf-PEI/siRNA polyplexes, lung function of animals was determined by PenH assay. The instrument setup is shown in Figure 3, and animals stay in a small chamber which is connected to a sensor that detects pressure changes in chamber and to a nebulizer that delivers the aerosol of a drug. We administrated increasing concentration of beta-methacholine, a drug that can induce bronchoconstriction. If Tf-PEI/siRNA polyplexes show any toxicity which impair lung function, we would anticipate to observe stronger bronchoconstriction at lower methacholine concentration than in healthy and in asthmatic untreated mice. Results of the PenH assay indicated that Tf-PEI/siRNA have minimal toxicity especially compared to PEI.


Figure 2 Setup of intratracheal instillation of siRNA polyplexes in anesthetized animals.


In the end, we harvested lung cells from animals and investigated what kind of cells took up most of the siRNA polyplexes. Results indicated that Tf-PEI could selectively deliver siRNA to activated T cells in asthmatic animals.

The importance of this study is that we demonstrated feasibility to selectively deliver siRNA with Tf-PEI to activated T cells in experimental asthma model. Although there are still more studies needed such as determination of therapeutic effects of administration of GATA-3 siRNA and evaluation of long-term biocompatibility of repeated doses of polyplexes, this study contributes the most important groundwork for targeting T cells in the lung as therapeutic approach for novel asthma therapy.