Chlamydia trachomatis (C. trachomatis) is a gram-negative bacterium that preferentially infects epithelial cells of the genital tract and causes the most common sexually transmitted bacterial infection in the world1. Unfortunately, about 80% of chlamydial infections in women are asymptomatic or with minimal symptoms, but if left untreated, the infection can lead to pelvic inflammatory disease, tubal infertility, ectopic pregnancy, premature delivery, and increased risk of developing cervical carcinoma. Furthermore, chlamydia infection can be passed to exposed newborns during birth resulting in conjunctivitis and possibly interstitial pneumonia2. The infection can also affect men, but it usually appears symptomatic and manifests as urethritis, and if left untreated, the infection can lead to epididymitis and proctitis1.

C. trachomatis is an obligate intracellular bacterium with two distinct forms, the infectious elementary body (EB) and the replicative reticulate body (RB) during its life cycle. Pathogenesis of chlamydia infection in the female genital tract begins with initial binding of EB to genital epithelial cells, and is followed by contiguous endocytosis through a membrane-bound compartment, inclusion3. After internalization, inclusion helps EB to rapidly escape the host endo-lysosomal pathway to avoid being degraded by the host defense system. At the same time, EB accomplishes the transformation into RB and begins to initiate bacterial protein synthesis. Newly synthesized inclusion membrane proteins assist the replication of RB by collecting and supplying nutrients from the host’s golgi3. As RB propagates and accumulates, the life cycle enters the late phase, in which late-phase effectors and EB effectors are being synthesized and the differentiation of new EB from RB is accomplished shortly afterwards. Eventually, newly produced EB leaves the host cells via extrusion (a process where a cell exports large particles or organelles through its cell membrane to the outside) or lysis to establish future infections3.

C. trachomatis is found to be able to infect various cell types in vitro and uses several receptors for binding to the host cells4. Initial binding of chlamydia starts with a primary reversible electrostatic interaction between EB and the host cell’s heparan sulfate receptor, followed by an irreversible secondary binding to other possible receptors such as the platelet derived growth factor receptor-β (PDGFR-β)5. Elwell et al. have shown that PDGFR-β knockdown with RNA interference decreased cell-associated bacteria by 50%. PDGFR plays an important role in vascular development and a mouse model has indicated that deletion of PDGFR-α or PDGFR-β causes no vascular defects but deletion of both disrupted the vascular development in yolk sac6. Further studies reported that an FDA-approved PDGFR inhibitor, imatinib, for the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia and gastrointestinal stromal tumors demonstrated minor adverse effects7. Therefore, knocking down PDGFR-β alone as a local therapy is expected to have negligible side effects. As a result, PDGFR-β can be potentially utilized as a therapeutic target to prevent and minimize chlamydia infection of host cells5.

Autophagy is a self-degradative process for providing energy under stressed conditions, degrading and recycling long-lived proteins and damaged organelles as well as playing an important role in eliminating intracellular pathogens (i.e. viruses and bacteria)8. Autophagy has been divided into three types: macroautophagy, microautophagy and chaperone-mediated autophagy. Among the three types, macroautophagy appears to play an important role in protecting cells against microbial infection9. Macroautophagy is composed of two subsequent stages, the initiation stage and the degradation stage9. During the initiation stage, a membrane called phagophore is invaginated from cell membranes and elongated to sequester components targeted for degradation, forming an enclosed double-membrane vesicle called autophagosome. This stage is subsequently followed by the degradation stage, during which the autophagosomes will then fuse with the acidic, enzyme-enriched lysosome, forming the degradative vesicle, autolysosome. During the fusion, only the outer layer of the double membrane of autophagosome fuses with lysosome while the inner layer becomes degraded, resulting in a single-membrane autolysosome. After that, the components inside of the autolysosomes are degraded into amino acids, fatty acids, nucleic acids and so forth for recycle and reuse8. Detecting and identifying the bacterial components that are attached to or inside of the cytoplasm of mammalian cells is a key process for initiating macroautophagy. Recognition of bacterial lipopolysaccharide by toll-like receptor 410, detection of bacterial peptidoglycan by NOD-like receptors11 as well as the identification of intracellular bacteria by sequestosome-1-like receptors12 are identified pathways for the initiation of autophagy in the defense against bacterial infection. Various regulatory proteins (e.g. Beclin-1 and class III phosphatidylinositide 3-kinase (VPS 34))13,14 are synthesized and recruited to initiate the nucleation of the phagophore, and as the phagophore elongates and grows into a complete autophagosome, a protein called microtubule-associated protein 1A/1B-light chain 3B (LC3B) starts to be synthesized from its precursor LC3A and becomes localized on autophagosomes13. As autophagy proceeds, autophagosomes begin to fuse with lysosomes and mature into degradative autolysosomes with LC3B internalized. At the same time, other regulatory proteins (e.g. tectonin beta-propeller repeat-containing protein-1 (TECPR-1) and UV radiation resistance-associated gene protein (UVRAG))14,15 are synthesized and recruited to promote the maturation of autolysosomes. Eventually, the pathogens as well as LC3B are degraded in autolysosomes by the enzymes and substances delivered from lysosomes. As a result, autophagy can be considered as an innate immune response against bacterial infection and studies have shown that autophagy can restrict intracellular growth of many bacteria such as streptococcus A16, mycobacterium tuberculosis17, listeria monocytogenes18, and C. trachomatis19.

Although chlamydial infection can be easily managed by macrolides or tetracyclines, the constant recurrence, the potential to develop antibiotic resistance20, the safety of antibiotic use during pregnancy21,22,23 and the common systemic side effects of antibiotics24,25 are always problems and concerns encountered in the healthcare setting. In an effort to control the prevalence of chlamydia, various screening programs have been established in different countries around the world. However, new cases and recurrent cases still pose a challenge in disease control2. Therefore, a safe non-antibiotic based therapy needs to be developed to provide alternative treatment choices for physicians and patients. C. trachomatis primarily targets epithelial cells as the first step to establish genital infection and forms inclusions to escape the endo-lysosomal degradation pathway. These two conditions predispose the genital epithelial cells with higher vulnerability to C. trachomatis compared to other immune cells in the genital tract. As a result, a therapy that can reduce bacterial binding to epithelial cells and induce autophagy in infected epithelial cells for the elimination of intracellular bacteria would be beneficial for combating the infection.

The use of small interfering RNA (siRNA) as a gene therapy to combat sexually transmitted infections has gained a lot of success during the past decade26,27,28,29. As a result, we propose a novel combination therapy involving the application of a siRNA-polyethylenimine-encapsulated nanoparticle fabricated with poly(lactic-co-glycolic acid)-polyethylene glycol (siRNA-PEI-PLGA-PEG NP) for the knock down of PDGFR-β expression and the simultaneous induction of autophagy as a strategy to prevent/reduce sexually transmitted chlamydia infection in women. This therapy can help prevent/reduce the acquisition and recurrence of chlamydia infection when used topically in the vagina prior to sexual intercourse. Knocking down PDGFR-β will prevent/reduce chlamydia entry into target host cells and inducing autophagy by encapsulating a cationic polymer PEI will help degrade the intracellular pathogens that have already invaded as mentioned above. This topical siRNA-based therapy will work locally to minimize the systemic side effects such as those caused by oral administration of antibiotics and the use of siRNA is associated with little to no development of resistance in bacterial cells.

The application of NP in pharmaceutical sciences has gained a lot of interests during the past three decades due to its advantages in promoting drug delivery. The diverse composition of NP with polymers, lipids and other materials improves bioavailability, biocompatibility and achieves sustained or stimuli-responsive drug release profiles, targeted drug delivery, enhanced pharmacokinetics, barrier penetration and so forth30,31,32,33,34,35,36.

Among all the biomaterials, PLGA is the most attractive and extensively used polymer for the development of intravaginal NP formulations. PLGA is a FDA-approved polymer that has been widely used in the preparation of NP due to its attractive properties including biodegradability, biocompatibility and versatility for encapsulating both small molecules and large molecules, capability for sustained drug release and possibility for surface modifications37. PLGA NP is highly biocompatible because PLGA can naturally degrade into lactic acid and glycolytic acid via ester bond hydrolysis within the body38. These two monomers can either enter the tri-carboxylic acid cycle for further breakdown into carbon dioxide and water or remain unchanged, and subsequently eliminated from the body39,40. Woodrow et al. successfully achieved intravaginal gene silencing in mice using siRNA-loaded PLGA NPs41. The use of PLGA NP was safe in vivo without triggering any immune responses41. Currently, one PLGA-based NP product (Eligard®) has been approved by the FDA for treating prostate cancer42.

Even though the use of PLGA NPs is safe and effective in gene knockdown, the mucus penetration ability of PLGA NPs was largely hindered by the hydrophobic interaction between the polymers and mucin fibers. In order to improve this, Hanes et al. have modified the hydrophobic PLGA NP with a dense coating of low molecular weight PEG resulting in significant improvement in mucus penetration36. Moreover, the addition of PEG to the system also improves the stability of NP in complex physiological environments by reducing their interactions with proteins and small molecules43. PEG is a FDA-approved polymer and its application is safe in humans and has been used in many FDA-approved medications including intravenous injections44,45.

Polyethylenimine (PEI) is a cationic polymer that can effectively condense hydrophilic siRNA through electrostatic interaction and facilitate effective encapsulation of siRNA into NPs46. Studies have shown that the use of PEI can improve the encapsulation efficiency of siRNA from 43% to 86%46. Moreover, like all cationic polymers potentially, PEI is capable of inducing autophagy in mammalian cells47. Chia-wei et al. have previously reported that branched PEI (25 K) was capable of inducing autophagy in mammalian cells. However, the use of PEI as a therapeutic therapy for promoting autophagy is largely limited by its cytotoxic effects, which are largely attributed to the permeabilization of plasma membranes48, decrease of nuclear size, decrease of lysosomal mass/pH, the permeabilization of mitochondrial membrane49 and induction of apoptosis and necrosis47.

As a result, we developed a siRNA-PEI-PLGA-PEG NP formulation to knock down PDGFR-β and promote autophagic flux in host cells simultaneously as a defensive strategy against C. trachomatis infection. The use of NP not only efficiently delivers siRNA into target cells but also reduces the cytotoxicity of PEI without compromising its ability in promoting autophagy.