In vitro release of plasmid DNA from oligo(poly(ethylene glycol) fumarate) hydrogels.

This research investigates the release of plasmid DNA in vitro from novel, injectable hydrogels based on the polymer oligo(poly(ethylene glycol) fumarate) (OPF). These biodegradable hydrogels can be crosslinked under physiological conditions to physically entrap plasmid DNA. The DNA release kinetics were characterized fluorescently with the PicoGreen and OliGreen Reagents as well as through the use of radiolabeled plasmid. Further, the ability of the released DNA to be expressed was assessed through bacterial transformations. It was found that plasmid DNA can be released in a sustained, linear fashion over the course of 45-62 days, with the release kinetics depending upon the molecular weight of the poly(ethylene glycol) from which the OPF was synthesized. Two formulations of OPF were synthesized from poly(ethylene glycol) of a nominal molecular weight of either 3.35K (termed OPF 3K) or 10K (termed OPF 10K). By the time the gels had completely degraded, 97.8+/-0.3% of the initially loaded DNA was recovered from OPF 3K hydrogels, with 80.8+/-1.9% of the initial DNA retaining its double-stranded form. Likewise, for OPF 10K gels, 92.1+/-4.3% of the initially loaded DNA was recovered upon complete degradation of the gels, with 81.6+/-3.8% of the initial DNA retaining double-stranded form. Experiments suggest that the release of plasmid DNA from OPF hydrogels is dominated by the degradation of the gels. Bacterial transformation results indicated that the DNA retained bioactivity over the course of 42 days of release. Thus, these studies demonstrate the potential of OPF hydrogels in controlled gene delivery applications.     

In vivo release of plasmid DNA from composites of oligo(poly(ethylene glycol)fumarate) and cationized gelatin microspheres.

Composites of cationized gelatin microspheres (CGMS), crosslinked with either 3 mM or 6 mM glutaraldehyde solution, and a novel hydrogel material, oligo(poly(ethylene glycol)fumarate) (OPF) were fabricated and investigated toward prolonging the release of plasmid DNA in vivo relative to the constituent materials. The composites and constituent materials were investigated in a subcutaneous murine model to assess the release of 125I-labeled plasmid DNA and 125I-labeled cationized gelatin in vivo. The time profiles of the radioactivity remaining were employed to compare the profiles of DNA release and cationized gelatin degradation. Both composite formulations (incorporating either 3 mM or 6 mM CGMS) prolonged the bioavailability of plasmid DNA relative to both injected plasmid DNA solution and the respective non-embedded cationized gelatin microspheres. Injected plasmid DNA solution persisted in the subject for only 7-10 days, whereas the persistence of DNA from composites of OPF and either 3 mM or 6 mM CGMS extended to at least day 42. The 3 mM and 6 mM CGMS each increased the persistence of DNA slightly, relative to injection of DNA solution, to between 28 and 35 days. Interestingly, the release profile of plasmid DNA from composites was not significantly different from the release of DNA from OPF alone. The release of plasmid DNA from the composites was in accord with the degradation of the microspheres within the OPF. These results show that composites of OPF and cationized gelatin microspheres are able to prolong the availability of plasmid DNA in vivo relative to cationized gelatin microspheres alone and provide a promising candidate material for the sustained, controlled release of plasmid DNA.     

In vitro release of the mTOR inhibitor rapamycin from poly(ethylene glycol)-b-poly(epsilon-caprolactone) micelles.

An injectable formulation of rapamycin was prepared using amphiphilic block co-polymer micelles of poly(ethylene glycol)-b-poly(epsilon-caprolactone) (PEG-PCL). Drug-loaded PEG-PCL micelles were prepared by a co-solvent extraction technique. Resulting PEG-PCL micelles were less than 100 nm in diameter and contained rapamycin at 7% to 10% weight and >1 mg/mL. PEG-PCL micelles released rapamycin over several days, t50% 31 h, with no burst release; however, physiological concentrations of serum albumin increased the release rate 3-fold. Alpha-tocopherol, vitamin E, was co-incorporated into PEG-PCL micelles and increased the efficiency of rapamycin encapsulation. The addition of alpha-tocopherol also slowed the release of rapamycin from PEG-PCL micelles in the presence of serum albumin, t50% 39 h.     

Poly-D,L-lactide-co-poly(ethylene glycol) microspheres as potential vaccine delivery systems.

Adjuvants aimed at increasing the immunogenicity of recombinant antigens remain a focus in vaccine development. Worldwide, there is currently considerable care for the development of biodegradable microspheres as controlled release of vaccines, since the major disadvantage of several currently available vaccines is the need for repeated administration. Microspheres prepared from the biodegradable and biocompatible polymers, the polylactide (PLA) or polylactide-co-glycolide (PLGA), have been shown to be effective adjuvants for a number of antigens. This review mainly focuses on polylactide-co-poly(ethylene glycol) (PELA) microspheres adjuvant as vaccine delivery systems by summarizing our and other research groups' investigation on properties of the microspheres formulation encapsulating several kinds of antigens. The results indicate that compared with the commonly used PLA and PLGA, PELA showed several potentials in vaccine delivery systems, which may be due to the block copolymer have its capability to provide a biomaterial having a broad range of amphiphilic structure. PELA microspheres can control the rate of release of entrapped antigens and therefore, offer potential for the development of single-dose vaccines. The PELA microspheres have shown great potential as a next generation adjuvant to replace or complement existing aluminum salts for vaccine potential. The review mainly aims to promote the investigation of PELA microspheres adjuvant for antigens for worldwide researcher.     

In vitro release of transforming growth factor-beta 1 from gelatin microparticles encapsulated in biodegradable, injectable oligo(poly(ethylene glycol) fumarate) hydrogels.

This research investigates the in vitro release of transforming growth factor-beta1 (TGF-beta1) from novel, injectable hydrogels based on the polymer oligo(poly(ethylene glycol) fumarate) (OPF). These hydrogels can be used to encapsulate TGF-beta1-loaded-gelatin microparticles and can be crosslinked at physiological conditions within a clinically relevant time period. Experiments revealed that OPF formulation and crosslinking time may be adjusted to influence the equilibrium swelling ratio, elastic modulus, strain at fracture, and mesh size of these hydrogels. Studies with OPF-gelatin microparticle composites revealed that OPF formulation and crosslinking time, as well as microparticle loading and crosslinking extent, influence composite swelling. In vitro TGF-beta1 release studies demonstrated that burst release from OPF hydrogels with a mesh size of 136 A was approximately 53%, while burst release from hydrogels with a mesh size of 93 A was only 34%. For hydrogels with a large mesh size (136 A), encapsulation of loaded gelatin microparticles allowed burst release to be reduced to 29-32%, depending on microparticle loading. Likewise, final cumulative release after 28 days was reduced from 71% to 48-66% by encapsulation of loaded microparticles. However, inclusion of gelatin microparticles within OPF hydrogels of smaller mesh size (93 A) was seen to increase TGF-beta1 release rates. The equilibrium swelling ratio of the microparticle component of these composites was shown to be greater than the equilibrium swelling ratio of the OPF component. Therefore, increased release rates are the result of disruption of the polymer network during swelling. These combined results indicate that the kinetics of TGF-beta1 release can be controlled by adjusting OPF formulation and microparticle loading, factors affecting the swelling behavior these composites. By systematically altering these parameters, in vitro release rates from hydrogels and composites loaded with TGF-beta1 at concentrations of 200 ng/ml can be varied from 13 to 170 pg TGF-beta1/day for days 1-3 and from 7 to 47 pg TGF-beta1/day for days 6-21. Therefore, these studies demonstrate the potential of these novel hydrogels and composites in the sustained delivery of low dosages of TGF-beta1 to articular cartilage defects.     

BSA-PLGA微球制备及不同添加剂对包封率和体外释放的影响

目的:探讨不同添加剂及制备工艺对BSA-PLGA微球包封率及体外释放的影响.方法:采用复乳-溶剂挥发法制备BSA-PLGA微球,考察BSA投入量、内水相聚乙二醇浓度及外水相氯化钠浓度对微球包封率和体外释放的影响.结果:通过正交设计,采用优化工艺制备的微球包封率达到了(91.07±4.22)%,突释率为(10.54±3.84)%,可缓释28 d左右.结论:通过对制备工艺进行优化,引入添加剂可以制得包封率高、突释率低的BSA-PLGA微球.     

In vitro study of GDNF release from biodegradable PLGA microspheres.

Glial cell line-derived neurotrophic factor (GDNF) is a protein with potent trophic actions on dopaminergic neurons, which is under investigation as a therapeutic agent for the treatment of neurodegenerative disorders, including Parkinson's disease. The aim of this work was to develop GDNF-loaded microspheres, which could be implanted by stereotaxy in the brain and could offer an alternative strategy in the treatment of Parkinson's disease. A w/o/w extraction-evaporation technique was chosen to prepare protein-loaded microspheres. An in vitro release study of the protein was required to assess the retention of integrity and the performance of the microsphere formulation with regard to sustained release. In order to assess the in vitro release profile of the GDNF-loaded microspheres, a preliminary study was performed to select an appropriate buffer for GDNF stabilization, using experimental designs. GDNF was measured by both enzyme-linked immunosorbant assay (ELISA) and radioactivity using (125)I-GDNF. The GDNF-loaded microsphere release profile was assessed in a low continuous flow system, and showed a sustained release over 56 days of biologically active GDNF at clinically relevant doses.     

In vitro degradation and controlled release behavior of D,L-PLGA50 and PCL-b-D,L-PLGA50 copolymer microspheres.

Blank and bovine serum albumin (BSA)-loaded microspheres based on poly(lactic-acid-alt-glycolic acid) (D,L-PLGA50) and poly(epsilon-caprolactone)-b-poly(lactic-acid-alt-glycolic acid) (PCL-b-D,L-PLGA50) were successfully fabricated using water-in-oil-in-water (w/o/w) double-emulsion extraction/evaporation technique. In vitro degradation of the blank microspheres was characterized by techniques including nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The PCL-b-D,L-PLGA50 copolymer (Mn: number-average molecular weight, Mw: weight-average molecular weight, Mn=44800, Mw/Mn=MWD=1.24, epsilon-caprolactone (CL) %=20.4% in molar ratio) had similar rate of molecular weight reduction compared with the D,L-PLGA50 copolymer before 5 weeks of in vitro degradation. The BSA % loading efficiency of microspheres was mainly controlled by both block copolymer composition and macromolecular architecture, while the sequence structure and the molecular weight of copolymer had no apparent effect on it. Significantly, The PCL-b-D,L-PLGA50 copolymer microspheres showed good release profiles with a nearly constant release during 20-110 days.     

In vitro and in vivo degradation of double-walled polymer microspheres

By exploiting the phenomenon of phase separation, double-walled microspheres consisting of a core of one polymer surrounded by a coating of a second polymer were formed using a modified process of solvent evaporation. This paper discusses the characterization and in vitro and in vivo degradation of these microspheres made of two biodegradable polymers with poly(lactic acid) (PLA) as the external layer and poly( 1,3-bis(p-carboxyphenoxypropane)-co-(sebacic anhydride)) 20:80 (P(CPP:SA)20:80) as the inner core. The microspheres degraded in vitro were analyzed by Fourier-transform infrared (FTIR) spectroscopy, gel permeation chromatography (GPC), differential scanning calorimetry (DSC), and optical and scanning electron microscopy (SEM). The same methods were used to characterize the microspheres used in the in vivo study before intramuscular implantation. The tissue containing the microspheres was explanted and studied histologically by optical microscopy and SEM. The microspheres from both studies showed the same patterns of degradation, albeit at slightly different rates. The polyanhydride was hydrolyzed into oligomers first, with the PLA degrading more slowly, decreasing in molecular weight and increasing in fragility over the course of the study. The main difference between the two studies was that in vitro the inner core of degrading polyanhydride was trapped by the outer layer of PLA, even as long as 187 days while after only 72 days in vivo the polyanhydride had disappeared.     

Influence of process parameters on the protein stability encapsulated in poly-DL-lactide-poly(ethylene glycol) microspheres.

Glucose oxidase (GOD) has been encapsulated as a model protein within poly-DL-lactide-poly(ethylene glycol) (PELA) microspheres to evaluate the activity retention during microencapsulation process. This paper was aimed to investigate the effect of process parameters, such as the preparation method, the used matrix polymer with different compositions, the solvent system and the addition of stabilizer on the structural integrity and activity retention of encapsulated protein. The stability of the protein released during in vitro assay was also assessed. The obtained results showed that the solvent extraction/evaporation method based on the formation of double emulsion w(1)/o/w(2) benefited the activity retention compared with the phase separation method based on the formation of w/o(1)/o(2). And in the emulsion-evaporation system most of the protein activity was lost during the first emulsification procedure to form primary emulsion w(1)/o (ca. 28%) and the second emulsification procedure to form the double emulsion w(1)/o/w(2) (ca. 20%), in contrast to other processes occurring during microspheres preparation. The matrix polymer and the solvent system in the oil phase had an impressive impact on the activity retention, while the addition of gelatin in the internal aqueous phase resulted in no major reduction of activity loss. GOD release from PELA microspheres exhibited a triphasic profile, that is, the initial burst release during the first day, the gradual release over about 1 month, and then the second burst release. The encapsulation of GOD in PELA microspheres was effective in reducing its specific activity loss. Sixty-seven per cent of the initial specific activity retention was detected for the released GOD from microspheres formulation during 1 week of incubation, but nearly all the activity was lost for GOD in solution incubated under the same condition. SDS-PAGE results showed that, although the activity loss was detected, no rough changes of molecular weight of GOD was observed during encapsulation procedure and the initial days of incubation into the in vitro release medium.     

Poly(ethylene glycol) hydrogels formed by conjugate addition with controllable swelling, degradation, and release of pharmaceutically active proteins.

Hydrogels were formed by conjugate addition of polyethylene glycol (PEG) multiacrylates and dithiothreitol (DTT) for encapsulation and sustained release of protein drugs; human growth hormone (hGH) was considered as an example. Prior to encapsulation, the hGH was precipitated either by Zn2+ ions or by linear PEG, to protect the hGH from reaction with the gel precursors during gelation. Precipitation by Zn2+ ions yielded precipitates that dissolved slowly and delayed release from even highly permeable gels, whereas linear PEG yielded rapidly dissolving precipitates. To independently protect the protein and delay its release, linear PEG precipitation was adopted, and release control via modulation of the PEG gel mesh size was sought. By varying the molecular weight of the multiarm PEG acrylates, control over gel swelling and hGH release, from a few hours to a few months, could be obtained. Protein release from the swollen and degrading PEG-based gel networks was modeled as a diffusion process with a time-dependent diffusion coefficient, calculated from swelling measurements and theoretical mesh sizes. Release following zero-order kinetics was obtained by the counter influences of decreasing protein concentration and increasing protein diffusion coefficient over time.     

Simple enzymatic screening assay for ethylene glycol (ethane-1,2-diol) in serum

We present a novel method for the analysis of ethylene glycol (ethane-1,2-diol) in serum. This method employs the ability of glycerol dehydrogenase (EC 1.1.1.6) to oxidize ethylene glycol. Two measurements, five and 30 min after addition of enzyme, are used to circumvent interferences from endogenous glycerol and from propylene glycol (propane-1,2-diol). Ethanol does not interfere in the assay. The method requires commonly available equipment only, making it suitable for all emergency laboratories.     

Poly(ethylene glycol)-human serum albumin-paclitaxel conjugates: preparation, characterization and pharmacokinetics.

Paclitaxel has been found to be very effective against several human cancers, such as ovarian, breast and non-small cell lung cancer and has received marketing approval for metastatic cancers. One of main problems with its use is its poor solubility, which makes irritant solubilitazion agents necessary. In previous research we demonstrated that linkage to human serum albumin (HSA) was useful to increase the in vivo performance of paclitaxel. In this article, in order to improve stability and solubility of paclitaxel conjugate, we linked covalently a monomethoxy poly(ethylene glycol) (mPEG) chain to HSA. New thioimidate mPEG derivatives, highly reactive and stable, were used and two different conjugates (with PEG of molecular mass 2 or 5 kDa) were prepared, purified and characterized. The antitumor activity of the free drug and conjugates was tested on three different tumor cell lines. The PEG grafted conjugates maintained high cytotoxicity, similar to that of ungrafted conjugates, with efficient cell binding and internalization followed by release of the drug inside the cell. The changes in pharmacokinetics and distribution of radio-labelled conjugates were evaluated by i.v. administration to mice and compared with those of the free drug and ungrafted conjugates. The total clearance was reduced (from 3.6 ml/h for free drug to 2.9, 1.97 and 1.41 for ungrafted, 2 and 5 kDa PEG conjugates, respectively). Organ uptake was reduced, in particular by liver and spleen.     

Controlled degradation of low-fouling poly(oligo(ethylene glycol)methyl ether methacrylate) hydrogels

Degradable low-fouling hydrogels are ideal vehicles for drug and cell delivery. For each application, hydrogel degradation rate must be re-optimized for maximum therapeutic benefit. We developed a method to rapidly and predictably tune degradation rates of low-fouling poly(oligo(ethylene glycol)methyl ether methacrylate) (P(EG)_xMA) hydrogels by modifying two interdependent variables: (1) base-catalysed crosslink degradation kinetics, dependent on crosslinker electronics (electron withdrawing groups (EWGs)); and, (2) polymer hydration, dependent on the molecular weight (M_W) of poly(ethylene glycol) (PEG) pendant groups. By controlling PEG M_W and EWG strength, P(EG)_xMA hydrogels were tuned to degrade over 6 to 52 d. A 6-member P(EG)_xMA copolymer library yielded slow and fast degrading low-fouling hydrogels suitable for short- and long-term delivery applications. The degradation mechanism was also applied to RGD-functionalized poly(carboxybetaine methacrylamide) (PCBMAA) hydrogels to achieve slow (∼50 d) and fast (∼13 d) degrading low-fouling, bioactive hydrogels.

To tune degradation rates of low-fouling hydrogels, a 6-member P(EG)_xMA copolymer library with different electronics and hydration levels was developed.     

Injectable microfluidic hydrogel microspheres based on chitosan and poly(ethylene glycol) diacrylate (PEGDA) as chondrocyte carriers

Direct injection of chondrocytes in a minimally invasive way has been regarded as the significantly potential treatment for cartilage repair due to their ability to fill various irregular chondral defects. However, the low cell retention and survival after injection still limited their application in clinical transformation. Herein, we present chondrocyte-laden microspheres as cell carriers based on a double-network hydrogel by the combination of the chitosan and poly(ethylene glycol) diacrylate (PEGDA). The microfluidic technique was applied to prepare size-controllable chitosan/PEGDA hydrogel microspheres (CP-MSs) via the water-in-oil approach after photo-crosslinking and physical-crosslinking. The chondrocytes were laden on CP-MSs, which showed good cell viability and proliferation after long-term cell cultivation. The in vitro investigation further demonstrated that chondrocyte-laden CP-MSs were injectable and the cell viability was still high after injection. In particular, these cell-laden microspheres were self-assembled into a 3D cartilage-like scaffold by a bottom-up strategy based on cell–cell interconnectivity, which suggested that these injectable chondrocyte-laden microspheres showed potential applications as chondrocyte carriers for bottom-to-up cartilage tissue engineering.

Chitosan/PEGDA double-network hydrogel microspheres prepared by microfluidic method as chondrocyte carriers for bottom-up cartilage tissue engineering.     

Triggered release of siRNA from poly(ethylene glycol)-protected, pH-dependent liposomes

The ability of small interfering RNA (siRNA) to regulate gene expression has potential therapeutic applications, but its use is limited by inefficient delivery. Triggered release of adsorbed poly(ethylene glycol) (PEG)-b-polycation polymers from pH-dependent (PD) liposomes enables protection from immune recognition during circulation (pH 7.4) and subsequent intracellular delivery of siRNA within the endosome (pH ~5.5). Polycationic blocks, based on either poly[2-(dimethylamino)ethyl methacrylate] (31 or 62 DMA repeat units) or polylysine (21 K repeat units), act as anchors for a PEG (113 ethylene glycol repeat units) protective block. Incorporation of 1,2-dioleoyl-3-dimethylammonium-propane (DAP), a titratable lipid, increases the liposome's net cationic character within acidic environments, resulting in polymer desorption and membrane fusion. Liposomes encapsulating siRNA demonstrate green fluorescent protein (GFP) silencing in genetically-modified, GFP-expressing HeLa cells and glyceraldehyde-3-phosphate dehydrogenase (GAPD) knockdown in human umbilical vein endothelial cells (HUVEC). Bare and PD liposomes coated with PEG113-DMA31 exhibit a 0.16 ± 0.2 and 0.32 ± 0.3 fraction of GFP knockdown, respectively. In contrast, direct siRNA administration and Oligofectamine complexed siRNA reduce GFP expression by 0.06 ± 0.02 and 0.14 ± 0.02 fractions, respectively. Our in vitro data indicates that polymer desorption from PD liposomes enhances siRNA-mediated gene knockdown.     

Stability of bovine serum albumin complexed with PEG-poly(L-histidine) diblock copolymer in PLGA microspheres.

The aim of this study was to examine the stability of bovine serum albumin (BSA) in poly(DL-lactic acid-co-glycolic acid) (PLGA) microspheres upon addition of a new excipient, poly(ethylene glycol)-poly(L-histidine) diblock copolymer (PEG-PH). Poly(L-histidine) component can form an ionic complex with BSA under acidic conditions within a narrow pH range. To optimize the ionic complexation conditions for BSA with PEG-PH, the resulting complex sizes were monitored using the Zetasizer. PLGA microspheres containing BSA as a model protein were prepared by w/o/w double emulsion method. BSA stability in aqueous solutions and after release from PLGA microspheres was determined using circular dichroism (CD) spectroscopy for secondary structure analyses and fluorescence measurements for tertiary structure analyses. The release profile of BSA from the microspheres was monitored using UV spectrophotometry. The rate of PLGA degradation was monitored by gel permeation chromatography. The pH profile within microspheres was further evaluated by confocal microscopy using a pH-sensitive dye. Approximately 19 PEG-PH molecules and one BSA molecule coalesced to form an ionic complex around a pH range of 5.0-6.0. Plain BSA/PLGA and BSA/PEG-PH/PLGA microspheres had a mean size of 27-35 microm. PLGA microspheres with a BSA loading efficiency >80% were prepared using the double emulsion method. PEG-PH significantly improved the stability of BSA both in aqueous solutions and in PLGA microspheres. The release profiles of BSA from different formulations of PLGA microspheres were significantly different. PEG-PH effectively buffered the local acidity inside the microspheres and improved BSA release kinetics by reducing initial burst release and extending continuous release over a period of time, when encapsulated as an ionic complex. PLGA degradation rate was found to be delayed by PEG-PH. There was clear evidence that PEG-PH played multiple roles when complexed with BSA and incorporated into PLGA microspheres. PEG-PH is an effective excipient for preserving the structural stability of BSA in aqueous solution and BSA/PLGA microspheres formulation.     

Sustained in vitro activity of human albumin microspheres containing chlorhexidine dihydrochloride against bacteria from cultures of organisms that cause urinary tract infections.

The potential of chlorhexidine dihydrochloride (CH HCl) incorporated into human albumin microspheres to provide sustained activity in vitro against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa from quality controls and from cultures of organisms that cause urinary tract infections was investigated. CH HCl was entrapped into five different formulations of human albumin microspheres. A technique was developed to evaluate the antibacterial activity of these microspheres and of controls (unloaded microspheres or gel). CH HCl microspheres exhibited antibacterial activity over a period of 16 days. Similar results were obtained with microspheres suspended in a methocel gel, and their antibacterial activity also continued for about 16 days. Empty microspheres or gel media alone were ineffective. The release rates of CH HCl from human albumin microspheres coated onto catheters by use of different gel formulations were also determined. The microsphere formulations were found to provide sustained antibacterial activity even at a low drug concentration.     

Poly(ethylene glycol)-containing hydrogels in drug delivery.

The use of hydrogels as carriers for protein delivery has been a subject of significant recent research. In our recent work, we have shown that diffusion controlled delivery of proteins from hydrogels containing poly(ethylene glycol) (PEG) can be possible and controlled by the three-dimensional structure. In addition, a number of these hydrogel carriers are mucoadhesive and can be used for protein delivery. PEG star polymer gels have also been prepared by gamma-irradiation and have been used for protein delivery with and without molecular imprinting. The presence of a large number of functional groups in a small volume makes these polymers important for use in biological and pharmaceutical applications. PEG star polymer hydrogels were synthesized using gamma-irradiation and were characterized using swelling techniques. Equilibrium swelling studies were conducted to investigate the effects of molecular weight, number of star arms, concentration, and radiation dose.