The scientific community has a great interest in the use of biopolymers for the delivery of siRNA therapeutics. The biocompatibility, high tolerability and versatility of these biopolymers are the main driving forces behind this interest. Results obtained by different groups around the world are augmenting this approach and more efforts are made to push these technologies up to the level of clinical trials. However, and despite the huge number of publications on the effective gene knockdown using siRNA therapeutics in cell lines and to a lesser extent in animals, progress to clinical trials is still significantly retarded. According to clinicaltrials.gov currently only one phase I clinical trial based on biopolymer delivery platform is taking place using a CDP‐based delivery system loaded with a siRNA active ingredient, under the name of CALAA‐01 (more details about this delivery system are discussed in Section 5: Cyclodextrins (CDs)). Even more challenging, Roche and Novartis announced the termination of their efforts to discover and develop drugs through RNA interference, thus abandoning well‐funded research projects and alliances and cutting more than 4000 jobs. Worth mentioning, however, other pharma companies such as GlaxoSmithKline and Sanofi‐Aventis along with many medium size pharmaceutical companies still believe in the potential of siRNA to create a novel generation of medications and are striving to push the limits of current technologies to achieve satisfactory performance.
From a pharmaceutical point of view, the current most important unmet challenge is efficient delivery of siRNA cargo intracellularly to target cells, at sufficient doses needed for optimal effect, preferably administered systemically and with a sustained mode of action as the patient in a real word setting cannot tolerate frequent injections. In our point of view, two main points might help to meet these challenges:
Taking into account the biocompatibility and tolerability of biopolymers compared to synthetic polymers, we see greater potential of their future use for this sort of delivery purposes. Thus, development of the production processes of these biopolymers to become more reproducible, controlled and avoid batch‐to‐batch variation is of great importance.
The last decade witnessed many advances within this context and some polymers are available now at pharma grade, however, still much is needed to gain the acceptance and accreditation of health authorities to use these polymers.
Development of novel controlled delivery platforms that could be administered systemically is crucial. One of the driving forces for the success of any pharmaceutical product is patient compliance in a real clinical setting and local administration of repetitive injections is definitely not meeting this need. Combination of current technologies may pave the way to achieving such delivery system.
Table 2: Selected examples of in vivo studies for siRNA delivery based on biopolymers
Single local injection in testis, 2.5 mg siRNA/50 ml/testis, (final atelocollagen conc. was 0.5%)
orthotopic xenograft model in athymic nude mice
Significant tumor volume reduction after 21 days if injection compared to control
[22]
VEGF On days 0, 10, 20, and 30: 50 µl of VEGF siRNA‐atelocollagen complex were injected into tumor cells (VEGF siRNA:
1,5,10 µm. Final atelocollagen conc. was 1.75%)
Prostate cancer xenograft model in athymic nude mice
VEGF siRNA‐ atelocollagen complex significantly suppressed tumor growth.
effect was dose dependent
[21]
EZH2/
p110‐α
1:1 siRNA atelocollagen mixture, 50µg siRNA‐atelocollagen complex on days 3, 6 and 9 postinoculation were i.v. injected (final atelocollagen conc. was 0.05%)
Bone metastatic tumour model in male athymic nude mice i.v. injected (final atelocollagen conc.
was 0.05%)
Liver metastatic mouse model
Long‐lasting inhibition of tumor growth for 60 days after last dose
[25]
Bcl‐xL Local intratumoral:
13.3µg of Bcl‐xL siRNA‐atelocollagen complex / 50 µl buffer
(final atelocollagen conc. was 0.5%) Administered once per week for 4 weeks. CDDP (2 mg/kg) was administered via ip injection 2 days after the siRNA injection.
Intravenous:
100µg of Bcl‐xL siRNA‐atelocollagen complex / 200 µl buffer (final atelocollagen conc. was 0.05%)
CDDP (2 mg/kg) was administered via ip injection 1 day after the last injection of siRNA in each administration week
Prostate cancer xenograft model in athymic nude mice
Significant downregulation of Bcl‐xL production
Effective inhibition of tumor growth when combined with cisplatin
[23]
myostatin Local intramuscular:
Myostatin siRNA‐atelocoallagen complex (final concentration, 10 mM) single i.m.
injection (final atelocollagen conc. was 0.5%)
Intravenous:
Myostatin siRNA‐atelocoallagen complex (final concentration, 40 mM)
injected 4 times in 3 weeks (final atelocollagen conc. was 0.05%)
C57BL/6 mice Significant increase in muscle volume and weight within few weeks
[26]
Gelatin
VEGF 10, 20 or 40 μg of siRNA expression plasmid DNA in 10 μL PBS injected intratumorally
Xenograft C3H/He mice
marked reduction in vascularity accompanied the suppression of VEGF production in the transfected tumor
[37]
TGF‐β 100 µg/mouse Male C57BL/6
mice
significant decrease of the level of TGF‐β and α‐smooth muscle actin over‐expression, the collagen content of mice kidney, and the fibrotic area of renal cortex, in contrast to free pDNA injection.
[38]
HSP47 50 µg/mouse, injected once intratumorally
Male ICR mice single injection of HSP47 siRNA‐CGM suppressed collagen synthesis even after 14 days
[39]
HSP47 Not reported Male ICR mice Prolonged release of siRNA for 21 days, suppressing the expression of collagen and infiltration of macrophages.
[29]
Tbox21 10 µg of Tbx21 siRNA loaded onto 1 mg of freeze‐dried cationized gelatin microspheres, was injected subcutaneously in 0.2 ml of PBS once a week for three times
C3H/HeJ mice successful elongated suppression of T‐box 21 protein leading to significant improvement of hair growth
[31]
TNF‐α, CyD1
3 oral doses of 1.2 mg NiMOS/kg/dose Balb/c mice successful gene silencing with the aid of dual siRNA treatment suppressed expression of several pro‐
inflammatory cytokines
[41]
Cyclodextrin
EWS‐FLI1 2.5 mg/kg dose by low‐pressure tail‐vein injection, twice weekly beginning the same day as injection of tumor cells
murine model of metastatic Ewing’s sarcoma
only 20% of the mice showing any tumor growth compared with 90% to 100% in other control groups
Escalating i.v. doses of 3 and 9 up to 27 mg siRNA/kg
cynomolgus monkeys
Smaller doses were well tolerated, higher doses induced kidney toxicity, and to a less extent liver toxicity and a mild immune response. in Male C57 BL/6 mice
VEGF siRNA achieved a significant inhibitory effect relative to the vehicle alone and approximately a 3‐fold reduction in tumour volume relative to PBS.
[51]
Bim and PUMA
intravenous injections of 50 µg of anti‐
Bim siRNA complex or anti‐Puma siRNA complex or both per mouse
male CD1 mice Significant decrease in Bim and PUMA concentration in splenic cells
[53]
TTR Intravenous injection of (1 mg/kg) or (9 mg/kg) of siRNA complex
GL3 intratumoral or intravenous injection with doses of 10 μg and 50 μg,
Dextran EGFR 6.65 μg EGFR siRNA was injected intratumorally to each mouse three times for 5 days.
Xenograft female BALB/c nude mice
significant tumor growth inhibitory effect was observed on day 12 to 20
[71]
Chitosan
TNFα Single dose of 200 μg/kg either by
intraperitoneal injection or oral gavage C57BL/6 mice TNFα knockdown was higher via the oral route than i.p injection (up to 74% and 39%
One dose of 4 μg TAMRA‐labeled NC siRNA
Female Kunming mice
8–26 folds enhancement in the TAMRA‐NC siRNA distribution level in plasma upon using NPs.
[105]
EGFP Formulations were sprayed each day over 3 consecutive days (working concentration
of siRNA was 50 μg/ml)
EGFP‐transgenic mice (C57BL/6‐
Tg)
Significant inhibition of GFP expression in the bronchial epithelial cells
Significant suppression of tumor growth
[107]
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