Patent Application
20130089495
1. Field of the Invention
The present invention provides a method for screening the size of carrier for a subject in need.
2. Description of the Related Art
The convergence of nanotechnology and biomaterials has spawned nanoparticles1,2, which have been widely used in medical applications, including drug delivery3,4, tissue engineering5,6, and medical imagine7,8. Accordingly, the toxicity of nanoparticles must be fully characterized before any nanoscale system can be used safely and efficiently for medical applications. There has been an increase in the number of studies reporting that the physical and chemical properties of size, shape, surface charge and functional groups influence the biodistribution, accumulation, and excretion of nanoparticles9,10. In previous studies, investigators have controlled the size and shape of nanoparticles to manipulate their behavior and to achieve enhanced, targeted drug delivery11,12. Research has also demonstrated that particle size greatly affects the transport and fate of the particle itself13,14. However, a comprehensive and systematic evaluation of nanoparticle on their biodistribution and on biological host responses in a quantitative and unambiguous manner has not yet been published. In the present invention, we develop methods to characterize the size effect of nanoparticles in vivo and to study the biodistribution of these particles in clinically relevant models.
Accordingly, the invention provides a method for detecting nanoparticles (NPs) in vivo retention (such as by HPLC), and the NPs size-dependent distribution in different conditions in various organs.
One object of the present invention is to provide a method for screening the size of carrier for a subject in need, which can be used as a screening platform for drug carrier, in which the optimal size of carrier can be screened for the organ and/or tissue affected by the condition of the subject. To achieve these objectives, the present invention provides a method for screening the size of carrier for a subject in need, comprising: (a) providing a series of labeled carriers which have different sizes; (b) administering one of the series of carriers to a subject who suffers from a condition selected from organ dysfunction, inflammation, cancer formation or other injured or abnormal conditions; (c) monitoring biodistribution of the carrier of step (b) in said subject; (d) repeating steps (b) and (c) until all the series of carriers are administered and all the biodistribution of the series of carriers are monitored; and (e) determining the size of carrier for said subject in accordance with the retention amount of the series of carriers in the tissue and/or organ affected by the condition of said subject.
In a preferred embodiment, the series of carriers are composed of an organic or an inorganic material, such as polystyrene, poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), gelatin, fibrin, agarose, chitosan, liposome, hyaluronic acid (HA), poly (ethylene glycol) (PEG), poly(propylacrylic acid) (PPAA) and N-isopropylacrylamide (NIPAAm); or composed of a metal material, such as gold.
In a preferred embodiment, the series of carriers are nanoparticles having a size in the range of 0.1-1000 nm; more preferably, in the range of 1-500 nm; even more preferably, in the range of 20-500 nm.
In a preferred embodiment, each of the series of carriers is fluorescence-labeled, radio-labeled, iron oxide-loaded, labeled by other materials or by methods for detection of the nanoparticles.
In a preferred embodiment, each of the series of carriers is administered by systemic intravascular, intramuscular or subcutaneous injection, oral intake, inhalation, or local skin, anal or vaginal administration.
In a preferred embodiment, the biodistribution of each carrier is monitored through in vivo, ex vivo or in vitro imaging system; more preferably, the imaging system comprises bioluminescence images (including immunofluorescent imaging), X-ray, CT, MRI, NMR, HPLC, PET/SPECT, ultrasound, OCT or other imaging methods for detecting the carriers, particularly, the nanoparticles.
In a preferred embodiment, the organ affected by the condition is brain, and the size of carrier is in the range of 0.1-1000 nm; more preferably, in the range of 1-500 nm; even more preferably, in the range of 20-500 nm. In the case of mammals, particularly in mice, the best size of carrier is in the range of less than 100 nm.
In a preferred embodiment, the organ affected by the condition is skin, and the size of carrier is in the range of 0.1-1000 nm; more preferably, in the range of 1-500 nm; even more preferably, in the range of 20-500 nm. In the case of mammals, particularly in mice, the best size of carrier is in the range of less than 100 nm.
In a preferred embodiment, the tissue affected by the condition is muscle, and the size of carrier is in the range of 0.1-1000 nm; more preferably, in the range of 1-500 nm; even more preferably, in the range of 20-500 nm. In the case of mammals, particularly in mice, the best size of carrier is in the range of less than 100 nm.
In a preferred embodiment, the organ affected by the condition is liver or spleen, and the size of carrier is in the range of 0.1-1000 nm; more preferably, in the range of 1-500 nm; even more preferably, in the range of 20-500 nm. In the case of mammals, particularly in mice, the best size of carrier is 20-500 nm.
In a preferred embodiment, the organ affected by the condition is lung, and the size of carrier is in the range of 0.1-1000 nm; more preferably, in the range of 1-500; even more preferably, in the range of 20-500 nm. In the case of mammals, particularly in mice, the best size of carrier is in the range of larger than 100 nm.
In a preferred embodiment, the organ affected by the condition is kidney, and the size of carrier is in the range of 0.1-1000 nm; more preferably, in the range of 1-500 nm; even more preferably, in the range of 20-500 nm. In the case of mammals, particularly in mice, the best size of carrier is in the range of larger than 20 nm.
Different nanoparticle properties, such as shape and surface charge, have been investigated to understand how to enhance the efficacy of nanoparticles in biomedical applications. However, there has not been a comprehensive study characterizing the size-dependency of nanoparticle biodistribution under different pathophysiologic conditions. Our study with fluorescent polystyrene nanoparticles revealed a size-dependent biodistribution of the nanopartieles that had been intravenously injected into normal mice. Further investigation showed that systemic inflammation induced by lipopolysaccharide changed the retention of the nanoparticles and led to redistribution in vital organs. Interestingly, we also observed a time-dependent distribution profile of the nanoparticles in a localized inflammatory hindlimb ischemia model. This model was validated by intravenous injection of polylactic-co-glycolic acid) (PLGA) nanoparticles that circulated into the ischemic areas. These unprecedented results show the importance of considering size when designing nanoparticles for use in nanoscale therapeutics and diagnostics.
Cells (A549 cells, A2058 cells, AFSCs and hMSCs) were seeded on to 12-well culture plates at a density of 2.6×104/cm2 in 1 ml total medium per well and allowed to adhere. 100 ul of 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) reagent (5 mg/ml 1XPBS) were added into each well and incubated for 2 hours at 37° C. After medium removal, 0.6 ml of DMSO was used to lyse cells and dissolve the formazan. The supernatant was collected and distributed into a 96-well plate at 0.2 ml each well for analysis. The absorbance was measured using ELISA reader (SpectraMax 340PC384, Molecular devices, USA) at 570 nm.
The National Cheng Kung University Animal Care and Use Committee and the National Laboratory Animal Center approved all animal research procedures. FVB and nude mice of either sex (6 to 8 weeks, weight 22±0.6 g) were purchased from the National Laboratory Animal Center.
Quantification and imaging of nanoparticle biodistribution analysis
Fluorescent carboxylated polystyrene latex bead nanoparticles with uniform diameters of 20, 50, 100, 200, and 500 nm (Invitrogen or Polyscience) were used to investigate the biodistribution and retention of nanoparticles after intravenous injection into mice. These nanoparticles were non-degradable, thus excluding resorption as a variable. Nanoparticles were quantified by high-performance liquid chromatography (HPLC, Jasco, Essex, UK). Fluorescence microscopy and an in vivo fluorescence imaging system (IVIS 200, Caliper Life Sciences, Massachusetts, USA) were used to observe the nanoparticle biodistribution in the tissues and organs.
To quantify the nanoparticle retention, normal, healthy, mice were anesthetized with Zoletil (50 mg/kg; Virbac, France) and Rompun (0.2 ml/kg; Bayer Healthcare, Germany), and injected with one of the five sizes of nanoparticles through the jugular vein (150 μl/mouse). Mice were returned to their cages and received a normal diet and water for 4 hours. Major organs and tissues, including the brain, heart, lungs, liver, spleen, kidneys, skin, fat and blood, and urine, were harvested. These harvested tissues, organs and urine were digested in 0.5 or 3 ml of 1 M potassium hydroxide
(KOH) solutions at 60° C. overnight, depending on the sample. The total volumes of the brain, heart, lung, liver, spleen, kidney, blood, skin and fat were digested in a 0.5 ml volume. Due to the size of the liver, 3 ml of a KOH solution was required for complete digestion. All of the samples were then mixed with 0.5 ml of o-xylene for fluorescein extraction by sonication for 1 min and placed into a 60° C. oven for 15 minutes. The samples were vortexed and incubated at 60° C. for 5 min; this step was repeated twice. For the urine sample, 0.5 ml of xylene was added directly without KOH digestion. The preparation of these samples then followed the procedures described previously. Finally, all of the samples were centrifuged for 30 minutes at 14,000 RPM, and the supernatants were analyzed by HPLC.
HPLC standards were measured by sampling 10, 40, 80, 160, and 200 μg of 20, 100, 200, and 500 nm nanoparticle solutions and 12.5, 25, 75, 100, and 150 μg of 50 nm nanoparticle solutions. The extraction procedures for nanoparticle standards were identical to the protocol described above. The relative amount of nanoparticle retention in each sample was calculated using the calibration standard curves.
Mice were anesthetized by injecting Zoletil (50 mg/kg; Virbac, France) and Rompun (0.2 ml/kg; Bayer Healthcare, Germany) before surgery was performed. For the systemic inflammation model, lipopolysaccharide (LPS, 5 mg/kg; Sigma, USA) was injected into the mice through the tail vein, followed with intravenous injection of nanoparticles after 24 hours. After four hours, mouse tissues and organs were harvested for sample preparation, as described above.
The hindlimb ischemia-reperfusion model was produced by ligating the right femoral artery of the unilateral right leg for 1 hour using a surgical suture. The sutures were then released for reperfusion for 6 hours, 1 day, or 3 days. Hindlimb blood flows were measured by laser Doppler (O2C flow meter, LEA Medizintechnik, Giessen, Germany) before and after surgery to confirm vessel occlusion. After 6 hours, 1 day and 3 days after reperfusion, the blood flow rates of both the injured leg (ischemic, right side) and the normal leg (nonischemic, left side) were measured, and different-sized nanoparticles were administered in the same procedure as outlined above. The muscles of both legs were harvested 4 hours after the nanoparticle injection. Samples were prepared and analyzed in the same procedure outlined above.
Poly(lactic-co-glycolic acid) (PLGA) was dissolved in 5 ml of acetone at a final concentration of 10 mg/ml. Ethanol/H2O (50/50, % v/v) solution was added dropwise (1 ml/min) to the PLGA solution using a tubing pump and stirred at 400 RPM until turbid. After 5 minutes of additional stirring, the suspension was transferred into a glass beaker containing 20 ml of 1 mM polyethylenimine (PEI, Sigma) solution and homogenized at low speed for 20 minutes as previously described25. The solution was filtered through a 0.22 μm membrane. The produced nanoparticles were washed three times with deionized water. The functional group of QD-COOH was linked to the NH2-terminated groups of PLGA NPs by adding 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). The surface morphology of PLGA NPs and PLGA-QD NPs, as shown in
Results are presented as the mean ±SEM. Statistical comparisons were performed with Student's t test. A probability value of p <0.05 was considered statistically significant. There were at least 6 animals in each group, unless specified.
To characterize the size-dependent effects of nanoparticles, commercially available 20, 50, 100, 200 and 500 nm fluorescent polystyrene nanoparticles were acquired. The nanoparticle sizes and shapes were confirmed by transmission electron microscopy, which showed uniform size distribution and consistent spherical morphology (
As depicted in
Nanoparticles are well-known for their short half-lives within the circulation and their rapid accumulation (within hours) in target tissues or organs9. To verify this biodistribution 3 mg (150 μl stock volume) of nanoparticles was injected into the jugular vein of healthy FVB mice and allowed to circulate for 4 hours. At this point, organs were collected for imaging with an in vivo imaging system (IVIS) or for sample preparation as outlined above for the HPLC analysis. IVIS images showed that the nanoparticles, regardless of size, were present in all of the vital organs, including the heart, lungs, liver, spleen, and kidneys (
When the biodistribution of the nanoparticles was analyzed by a weight-to-weight ratio of nanoparticles to organs, nanoparticles were revealed to be more evenly distributed by nanoparticle density among the heart, lungs, liver, spleen, and kidneys (
Detailed inspection of the HPLC quantification of brain nanoparticle retention revealed contradictory results. The IVIS images only showed the presence of nanoparticles smaller than 100 nm (
Most of the larger nanoparticles (100 nm or greater) were retained in the vital organs and the blood, and less than 20% of the smaller nanoparticles (below 100 nm) were recovered from our samples (
Drug nanocarriers have been designed to target tissues under specific disease conditions2,4,10,11. A system under a disease condition responds differently to foreign bodies than a normal, healthy, system. The diseased system responds differently by altering microenvironmental conditions, varying cell behavior, and using signal transduction pathways that result in specific responses against the foreign bodies17-19. Thus, it is crucial that the uptake and distribution of nanoparticles be fully characterized in the diseased state of the model for the drug delivery system. To investigate whether a change in pathophysiologic conditions affects the biodistribution of nanoparticles, the same procedure was repeated as outlined above (
In the heart, lungs, liver, spleen, and kidney, larger nanoparticles were evenly distributed (
Tissue sections, IVIS imaging, and HPLC analysis also confirmed that more nanoparticles were retained in the brain and spleen but that fewer were retained in the lungs and liver of LPS-treated mice (
As mentioned above, additional tissues were analyzed. The IVIS images showed that larger nanoparticles were detected in the skin and muscle (
In contrast, local injuries may be not accompanied by the same heightened systemic response by the body as with systemic inflammation. To investigate whether changes in the microenvironmental conditions surrounding the diseased tissue altered the nanoparticle kinetics, a hindlimb ischemia-reperfusion model was performed as described previously23 with some modifications. After femoral arterial ligation for 1 hour, the artery was allowed to reperfuse for 6 hours, 1 day, or 3 days (
We used poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) to confirm our results in a relevant clinical setting. PLGA NPs have been well-established and well-characterized in several drug delivery systems3,24,25. Previous studies have demonstrated success in therapeutic treatments, and drug-containing PLGA NPs are also approved by the US Food and Drug Administration for clinical use. Using PLGA
NPs, the drug nanocarrier design considerations were tested with more relevant biomaterial than fluorescent polystyrene NPs, allowing us to generalize design principles for all drug nanoparticles, regardless of the material. We theorized that a PLGA NP drug delivery carrier smaller than 100 nm in diameter would result in greater retention in the muscle of a hindlimb ischemia-reperfusion model. We synthesized 80 and 300 nm PLGA NPs conjugated with quantum dots (PLGA-QD NPs;
In the present study, the size-dependent biodistribution of nanoparticles ranging from 20 to 500 nm was systematically characterized in a mouse model. Our results indicate that most of the vital organs retained the nanoparticles in a size-dependent manner. Larger nanoparticles, particularly those with a diameter greater than 100 nm, were more likely to be distributed in the vital organs. Small nanoparticles, with a diameter of less than 100 nm, were mostly retained in the peripheral tissues or were excreted via the urine. Additionally, systemic inflammation and local hindlimb ischemia altered the biodistribution pattern to allow large nanoparticles to be retained in the vital organs and in the peripheral tissues. Our results were validated by the injection of a nanoparticles produced from an FDA approved material, PLGA. We consider that the comprehensive characterization of nanoparticle behavior in vivo presented in this study is important for nanomedicine design considerations. The conclusions drawn from our results should be taken into account when designing nanoparticles for intravenous drug delivery.
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| Number | Date | Country | |
|---|---|---|---|
| 61543400 | Oct 2011 | US |
